Cortistatin analogues and syntheses and uses thereof

ABSTRACT

Provided herein are compounds of Formula (A), (B), (C), (D) and (E), pharmaceutically acceptable salts, quaternary amine salts, and N-oxides thereof, and pharmaceutical compositions thereof. 
     
       
         
         
             
             
         
       
     
     Compounds of Formula (A), (B), (C), (D), and (E) are contemplated useful as therapeutics for treating a wide variety of conditions, e.g., including but not limited to, conditions associated with angiogenesis and with CDK8 and/or CDK19 kinase activity. Further provided are methods of inhibiting CDK8 and/or CDK19 kinase activity, methods of modulating the □-catenin pathway, methods of modulating STAT1 activity, methods of modulating the TGFβ/BMP pathway, methods of modulating HIF-1-alpha activity in a cell, and methods of increasing BIM expression to induce apoptosis, using a compound of Formula (A), (B), (C), (D), or (E). Further provided are CDK8 and CDK19 point mutants and methods of use thereof.

STATEMENT OF RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2014/072365 filed on Dec. 24, 2014, which is related to and claimspriority benefit of U.S. provisional patent application 61/920,674 filedDec. 24, 2013; U.S. provisional patent application 61/935,240 filed Feb.3, 2014; and U.S. provisional patent application 61/993,329 filed May15, 2014, the entire contents of each of which is incorporated herein byreference.

BACKGROUND

The cortistatins are a group of anti-angiogenic steroidal alkaloidsfirst isolated in 2006 from the marine sponge Corticium simplex. See,e.g., Aoki, et al., JACS (2006) 128: 3148-9. From the date of isolationto the present, these natural products have been the subject of muchstudy, especially in the development of total syntheses and of newunnatural biologically active analogs. See, e.g., Aoki et al.,Bioorganic & Medicinal Chemistry (2007) 15: 6758-62. Mousseau et al.,Cell Host & Microbe (2012) 12: 97-108; Chen et al., Organic &Biomolecular Chemistry (2010) 8: 2900; Hardin et al., European Journalof Organic Chemistry (2010) 19: 3553. Thus, there is an active interestin the development of new cortistatin analogs and methods of theirpreparation.

SUMMARY OF THE INVENTION

Provided herein are new cortistatin analogs of Formula (A), (B), and(C), and pharmaceutically acceptable salts, quaternary amine salts, andN-oxides thereof, synthesized, in part, by reductive amination of aketone of Formula (B) to provide the aminated product of Formula (A), asdepicted in Scheme 1, optionally via an imine intermediate of Formula(C).

Further provided are new cortistatin analogs prepared by reduction ofthe ketone of Formula (B) to provide a C3-hydroxyl compound of Formula(D). Still yet further provided is a compound of Formula (E), preparedby substitution of the compound of Formula (D). Such compounds may alsobe converted to a compound of Formula (A) upon treatment with an amineunder suitable conditions.

It has been surprisingly found that the beta isomers of Formula (A),referred to as Formula (A-1), have been found to be equipotent, or morepotent, than cortistatin A at inhibiting CDK8 kinase activity and theproliferation of AML cells, and it has also been found that thecorresponding alpha isomers of Formula (A), referred to as Formula(A-2), are also very potent. Furthermore, it has been discovered thatcompounds of Formula (B) have been found active against the growth ofAML cell lines in culture and CDK8 kinase activity in cells.

Further provided are pharmaceutical compositions comprising acortistatin analogs of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, and a pharmaceutically acceptable excipient. Further providedare methods of use and treatment.

Thus, as further described herein, in one aspect, provided is a methodof preparing a compound of Formula (A), or a pharmaceutically acceptablesalt, quaternary amine salt, or N-oxide thereof; the method comprisingcontacting a compound of Formula (B), or a pharmaceutically acceptablesalt, provided R^(B1) and R^(B2) are not joined to form an oxo group;with an amine of formula HNR¹R², or salt thereof, under reductiveamination conditions.

In another aspect, provided is a method of preparing a compound ofFormula (D), or a pharmaceutically acceptable salt thereof; the methodcomprising contacting a compound of Formula (B), or a pharmaceuticallyacceptable salt, with a reducing agent, to provide a compound of Formula(D).

In another aspect, provided is a method of preparing a compound ofFormula (E), or a pharmaceutically acceptable salt thereof; the methodcomprising contacting a compound of Formula (D), or a pharmaceuticallyacceptable salt thereof, with a compound of formula R^(O)-LG, wherein LGis a leaving group, to provide a compound of Formula (E).

In another aspect, provided is a method of preparing a compound ofFormula (A), or a pharmaceutically acceptable salt, quaternary aminesalt, or N-oxide thereof; the method comprising contacting a compound ofFormula (E), wherein R^(O) is C(═O)R^(A), or a pharmaceuticallyacceptable salt thereof, with a compound of formula NHR¹R², to provide acompound of Formula (A).

In another aspect, provided are pharmaceutical compositions comprising acompound of Formula (A), (B), (C), (D), or (E), or a pharmaceuticallyacceptable salt, quaternary amine, or N-oxide thereof.

In another aspect, provided is a method of treating a conditionassociated with angiogenesis comprising administering to a subject inneed thereof a compound of Formula (A), (B), (C), (D), or (E), or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof,or a pharmaceutical composition thereof. In certain embodiments, thecondition is a diabetic condition, an inflammatory condition, maculardegeneration, obesity, atherosclerosis, or a proliferative disorder.

In yet another aspect, provided is a method of treating a conditionassociated with CDK8 and/or CDK19 kinase activity, comprisingadministering to a subject in need thereof a compound of Formula (A),(B), (C), (D), or (E), or a pharmaceutically acceptable salt, quaternaryamine, or N-oxide thereof, or a pharmaceutical composition thereof. Incertain embodiments, the condition is a proliferative disorder. Incertain embodiments, the proliferative disorder is cancer. In certainembodiments, the cancer is a hematopoietic cancer. In certainembodiments, the hematopoietic cancer is lymphoma. In certainembodiments, the hematopoietic cancer is leukemia. In certainembodiments, the hematopoietic cancer is multiple myeloma. In certainembodiments, the leukemia is acute myelocytic leukemia (AML). In certainembodiments, the proliferative disorder is a myeloproliferativeneoplasm. In certain embodiments, the myeloproliferative neoplasm isprimary myelofibrosis (PMF). In certain embodiments, the cancer is asolid tumor.

In yet another aspect, provided is a method of inhibiting CDK8 and/orCDK19 kinase activity in a cell comprising contacting a compound ofFormula (A), (B), (C), (D), or (E), or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof, or a pharmaceuticalcomposition thereof, with the cell.

In yet another aspect, provided is a method of modulating the β-cateninpathway in a cell comprising contacting a compound of Formula (A), (B),(C), (D), or (E), or a pharmaceutically acceptable salt, quaternaryamine, or N-oxide thereof, or a pharmaceutical composition thereof, withthe cell.

In yet another aspect, provided is a method of modulating STAT1 activityin a cell comprising contacting a compound of Formula (A), (B), (C),(D), or (E), or a pharmaceutically acceptable salt, quaternary amine, orN-oxide thereof, or a pharmaceutical composition thereof, with the cell.

In yet another aspect, provided is a method of modulating the TGFβ/BMPpathway in a cell comprising contacting a compound of Formula (A), (B),(C), (D), or (E), or a pharmaceutically acceptable salt, quaternaryamine, or N-oxide thereof, or a pharmaceutical composition thereof, withthe cell.

In yet another aspect, provided is a method of modulating HIF-1-A(HIF-1-alpha) activity in a cell comprising contacting a compound ofFormula (A), (B), (C), (D), or (E), or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof, or a pharmaceuticalcomposition thereof, with the cell.

In yet another aspect, provided is a method of increasing BIM expressionto induce apoptosis in a cell comprising contacting a compound ofFormula (A), (B), (C), (D), or (E), or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof, or a pharmaceuticalcomposition thereof, with the cell.

In any of the above recited methods, the method is an in vitro method oran in vivo method.

Further provided are CDK8 or CDK19 Tpr105 point mutants, and methods ofuse thereof. For example, in one aspect, provided is a method ofvalidating CDK8 and/or CDK19 kinase activity in a cell by contacting aCDK8 or CDK19 Trp105 point mutant and a cortistatin or cortistatinanalog. In another aspect, provided is a CDK8 Trp105 point mutant. Incertain embodiments, the CDK8 Trp105 point mutant has an amino acidsequence that a degree of homology to the amino acid sequence of SEQ IDNO: 1 of at least about 80%. Further provided is a protein that has adegree of homology to the amino acid sequence of SEQ ID NO: 1 of atleast about 80%. In yet another aspect, provided is a CDK19 Trp105 pointmutant. In certain embodiments, the CDK19 Trp105 point mutant has anamino acid sequence that a degree of homology to the amino acid sequenceof SEQ ID NO: 2 of at least about 80%. Further provided is a proteinthat has a degree of homology to the amino acid sequence of SEQ ID NO: 2of at least about 80%.

The details of one or more embodiments of the invention are set forth inthe accompanying Figures. Other features, objects, and advantages of theinvention will be apparent from the Detailed Description, the Examples,and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of cortistatin A and other knowncortistatins.

FIG. 2A and FIG. 2B depict an exemplary reductive amination of ketone 13to provide compounds 14A and 14B, and their corresponding N-oxides (FIG.2A) and the molecular structure of compound 14B, wherein thedimethylamine is beta (axial) (FIG. 2B).

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H,FIG. 3I, FIG. 3J, FIG. 3K, FIG. 3L, and FIG. 3M depict exemplarycompounds of the present invention. Additional compounds are furtherdescribed herein.

FIG. 4A and FIG. 4B depict the dose-dependent inhibition ofproliferation of AML cell lines MV4;11 (4A) and MOLM-14 (4B) upon 7-daytreatment with cortistatin A and the indicated analogs. Cells werepassaged and fresh compounds were added on day 3 (mean+/−standard error,n=3).

FIG. 5A and FIG. 5B depict the dose-dependent inhibition ofproliferation of AML cell line MV4;11 upon 3-day or 10-day treatmentwith the indicated cortistatin analogs. Cells were passaged and freshcompounds were added on days 3 and 7 (mean+/−standard error, n=3).

FIG. 6A and FIG. 6B depict the dose-dependent inhibition ofproliferation of AML cell line MOLM-14 upon 3-day or 10-day treatmentwith the indicated cortistatin analogs. Cells were passaged and freshcompounds were added on days 3 and 7 (mean+/−standard error, n=3).

FIG. 7 demonstrates cortistatin A potently and selectively inhibits CDK8module kinase activity in cells. CDK8 has been reported to inhibitinterferon gamma-stimulated phosphorylation of STAT1 S727. See, e.g.,Bancerek et al., Immunity (2013) 38:250-262. HepG2 cells were incubatedwith cortistatin A for 1 hour followed by the addition of humanrecombinant interferon-gamma (IFN-γ) for 1 hour. The cells were lysedand probed by western blot. As shown, cortistatin A potently inhibitedthe IFN-γ-stimulated increase in STAT1 pS727 without altering levels ofSTAT1 pY701 or total STAT1 and Actin.

FIG. 8A, FIG. 8B, and FIG. 8C demonstrate cortistatin A inhibitsproliferation of human cell lines derived from patients with a varietyof hematologic malignancies and bearing diverse oncogenic drivers. Thehematologic malignancies represented by these cell lines are: AML,including those derived from patients with myeloproliferative neoplasms(MPNs, UKE-1 and SET-2), T-ALL, B-ALL, CML, and MM. Oncogenic driversinclude MLL-fusions with FLT3-internal tandem duplications (FLT3-ITD)(MOLM-14, and MV4;11) and without FLT3-ITD (RS4; 11), JAK2V617F, andBCR-ABL. Also shown are JAK2V617F-expressing cell lines that have beensubjected to prolonged treatment with JAK1/2 inhibitor ruxolitinib(SET-2per, UKE-1per) as previously described. See, e.g., Koppikar etal., Nature (2012) 489:155-159. As shown, cortistatin A also inhibitedthe proliferation of these cells that persist in presence ofruxolitinib. Cells were passaged and fresh cortistatin A was added ondays 3 and 7 (mean+/−standard error, n=3).

FIG. 9 depicts the dose-dependent inhibition of proliferation ofmultiple myeloma cell line MM. 1S upon 10-day treatment with theindicated cortistatin analogs. Cells were passaged and fresh compoundswere added on days 3 and 7 (mean+/−standard error, n=3).

FIG. 10A, FIG. 10B, and FIG. 10C demonstrate that CDK8/CDK19 mediate theantiproliferative effects of cortistatin A by showing that alleles ofCDK8 and CDK19 render cells resistant to growth inhibition bycortistatin A (FIG. 10A) and resistant to CDK8 kinase inhibition bycortistatin A in vitro (FIG. 10B) and in cells (FIG. 10C).

FIG. 11A and FIG. 11B depict pharmacokinetic results for cortistatin A.FIG. 11A: Cortistatin A was administered IP in a 10% DMSO/phosphatebuffered saline at a pH6 and at 10 mg/kg to male CD-1 mice and serialblood plasma samples were collected and analyzed for the presence ofcortistatin A. As indicated, the C_(max) was 1.4 microM at 1 hour andthe calculated T ½ was 6.06 hours. Based on this PK study and the invitro potency of cortistatin A, it is predicted that cortistatin A maymaintain an efficacious dose in mice with at least 0.16 mg/kg once dailytreatment. FIG. 11B: Male CD-1 mice dosed with single IP injection of 1mg/kg cortistatin A in 20% hydroxypropyl-beta-cyclodextrin (HPCD). TheC_(max) was 762 nM at 30 minutes and the calculated T½ between 0.5-2 hwas 33 minutes.

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, and FIG. 12Gdemonstrate cortistatin A is effective in MV4;11 disseminated leukemiamouse model of AML. MV4;11 AML cells expressing mCherry, luciferase andpuromycin resistance gene were injected into the tail-vein ofimmunocompromised NOD-SCID-IL2Rcγ^(null) (NSG) mice. After 7 days,engraftment was documented by bioluminescence imaging and mice weretreated with vehicle (20% 2-hydroxypropyl β-cyclodextrin), 0.05 and 0.16mg/kg once daily for 15 days. Time after first treatment is indicated.FIG. 12A shows bioluminescent images of the mouse with the medianbioluminescence for each timepoint, indicating a lower disease burdenover time. FIG. 12B shows the mean bioluminescence±s.e.m., n=11,P<0.0001, determined by two-way ANOVA. FIG. 12C shows the Kaplan-Meiersurvival analysis (n=8, P<0.0001, log-rank test). FIGS. 12D-12F depictthe day 30 spleen weight (D) and percentage of MV4;11 cells(mCherry-positive) in the spleen (E) and femur bone marrow (F) are shownfor the mouse in each group with the highest, lowest, and median day 29bioluminescence. *, P<0.05, **, P<0.01, ***, P<0.001, determined byone-way ANOVA vehicle vs. treatment. Dotted lines in FIG. 12D mark therange within 1 standard deviation of mean for healthy 8-week old femaleNSG mice. FIG. 12G shows that the mean body weight did not decrease upontreatment with cortistatin A, suggesting that cortistatin A treatment istolerated. Mean body weight±s.e.m., n=11.

FIG. 13A and FIG. 13B, demonstrate cortistatin A is tolerated in healthyCD-1 mice. CD-1 mice were treated with the same dosing regimen as inFIG. 12 (once daily for 15 days) and body weight and complete bloodcount (CBC) were performed. FIG. 13A, Mean body weight±s.e.m., n=3. FIG.13B, CBC data collected 2 hours after the last dose on day 15. CBCanalysis indicates no significant differences between vehicle and 0.16mg kg⁻¹. RBC, red blood cells (×10⁶ cells/μl); HGB, hemoglobin (g/dl);HCT, hematocrit (%); MCV, mean corpuscle volume (fl); MCH, meancorpuscle hemoglobin (pg); MCHC, mean corpuscle hemoglobin (g/dl); PLT,platelets (×10⁵ platelets/μl); WBC, white blood cells (×10³ cells/μl);LYMPH, lymphocytes (×10³ cells/μl).

FIG. 14A, FIG. 14B, and FIG. 14C, demonstrate cortistatin A showspromise as a therapeutic in the treatment of primary myelofibrosis(PMF). The results are from a pilot study in a murine model of PMF. Inthis model, MPLW515L overexpression in hematopoietic stem cells leads tomyeloproliferation in vivo, with marked bone marrow fibrosis,splenomegaly, and megakaryocyte proliferation (See, e.g., Pikman et al.,PLoS Med. (2006) 3:e270). In this model, mice were transplanted withMPLW515L-transduced bone marrow. After 14 days to allow for engraftmentand development of severe MPN (including thrombocytosis, leukocytosis,and myelofibrosis), mice were randomized to receive vehicle orcortistatin A i.p. once daily. Sacrifice of 3 mice per group after 6doses of daily cortistatin A shows significant reduction of spleenweights at 0.31 mg/kg and 0.62 mg/kg (A). Macroscopic reduction ofsplenomegaly is shown in (C). Analysis of blood counts after 4 doses ofdaily treatment shows significant reduction of allele burden asreflected by GFP percentage in peripheral blood (the MPLW515L mutant isexpressed using a MSCV-IRES-GFP retrovirus) (B).

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E, demonstratescortistatin A can modulate Wnt/beta-catenin (A,B) and TGF-beta (C)pathways and can induce BCL2L11 expression (D and E) in cells. FIG. 15Aindicates cortistatin A inhibits Wnt/beta-catenin pathway-stimulatedreporter gene expression. HEK-293 cells stably transfected with aluciferase reporter under the control of several TCF binding sites(referred to as SuperTOPflash)(See, e.g., Xu, et al., Cell 2004, 116,883-895). Upon 24 hour treatment with either Wnt3A or 10 μM GSK3βinhibitor azakenpaullone to stimulate the Wnt/beta-catenin pathway,cortistatin A inhibited expression of luciferase as measured byluminescence (IC₅₀=1.5 nM, without affecting HEK-293 cell viability.Vertical bars=SEM, n=3. FIG. 15B indicates that cortistatin A inhibitsputative Wnt/beta-catenin response genes in the AML cell line MV4;11.MV4;11 cells were treated with cortistatin A for 24 h and geneexpression analysis and Gene Set Enrichment Analysis (GSEA) wasperformed. The GSEA revealed that cortistatin A downregulated a set ofgenes that are upregulated upon GSK3-beta inhibition. Since GSK3-betainhibition can stimulate beta-catenin driven transcription, these genesrepresent putative Wnt/beta-catenin pathway genes. FIG. 15C indicatescortistatin A (CA) dose-dependently inhibited TGF-beta-stimulatedSMAD2/3 phosphorylation. HaCat cells were treated with vehicle or CA for1 hour followed by TGF-beta for 1 hour. Cells were then washed, lysed,and analyzed by Western blot. FIGS. 15D and E indicate cortistatin Adose-dependently increases mRNA levels of BCL2L11 upon 24 h treatment ofMV4;11 and MOLM-14 cells.

FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D, depict binding ofcortistatin A to the CDK8/cyclin C ATP binding site. FIG. 16A depictsthe X-ray crystal structure of cortistatin A/CDK8/cyclin C ternarycomplex at 2.4 Angstrom resolution. FIG. 16B depicts the binding pocketshowing equisite shape complementarity. FIG. 16C depicts the key contactresidues between CDK8 and cortistatin A. The dotted line indicates anH-bond between the isoquinoline N of cortistatin A and the main chainamide N-H of CDK8. Electron density of cortistatin A also shown. FIG.16C also includes the structural alignment of ATP (cyan/orange, fromCDK2-ATP structure) with X-ray structure of cortistatin A (yellow) inCDK8. Note that Trp105 does not contact ATP. FIG. 16D depicts a closerview of the contact between Trp105 and the N,N-dimethyl group ofcortistatin A. As further described herein, a Trp105 mutation(replacement with Met) confers resistance to cortistatin A.

FIG. 17A and FIG. 17B, FIG. 17A depicts the synthesis of the C₃—N-oxideof cortistatin A upon exposure of cortistatin A to silica gel, incontrast to treatment of cortistatin A with triethylamine treated silicagel. FIG. 17B depicts the sensitivity of HUVECs to treatment withcortistatin A and cortistatin A N-oxide and shows that the 2 compoundsare equipotent. Growth inhibition after 96 hour treatment of HUVECs wasmeasured by fluorescence signal using Celltiter Blue (Promega) withvehicle (DMSO) representing maximal growth and 10 μM doxorubicinrepresenting maximal growth inhibition.

FIG. 18 demonstrates that cortistatin A is effective in SET-2subcutaneous xenograft model of JAK2V617F-driven proliferation. 1×10⁷SET-2 cells tumor cells in 50% matrigel were injected subcutaneouslyinto the flank of 8 to 12 week old female SCID Beige mice. Once tumorsreached an average size of 80-120 mm³, the mice were treated withvehicle or 0.16 mg kg⁻¹ daily for the duration of the study, resultingin 68% tumor growth inhibition (n=10, P<0.001, 2way ANOVA).

FIG. 19 depicts in vitro liver microsome metabolic stability assaydemonstrating the N-oxide of compound 14B demonstrates improvedstability.

FIG. 20 depicts the results of an in vitro liver microsome assaydemonstrating compound 14B undergoes N-monodemethylation at C₃ in humanliver microsomes to compound 24B.

FIG. 21A, FIG. 21B, FIG. 21C, FIG. 21D, and FIG. 21E, demonstrateequipotency of the N-oxide of compound 14B and compound 14B in celllines tested. Cells were passaged and fresh compounds were added on days3 and 7 (mean+/−standard error, n=3).

FIG. 22A and FIG. 22B, demonstrate alleles of CDK19 (FIG. 22A) and CDK8(FIG. 22B) render MV4;11 cells resistant to growth inhibition by theN-oxide of compound 14B (14BNO), indicating that CDK8/CDK19 mediate theantiproliferative effects of 14BNO.

FIG. 23 demonstrates that the N-oxide of compound 14B (14BNO) has anacceptable half-life and clearance rate upon mixing with pooled humanhepatocytes.

FIG. 24A, FIG. 24B, and FIG. 24C provides the results of apharmacokinetic study with compound (14A) formulated in 20%hydroxypropyl-beta-cyclodextrin in male CD-1 mice given a single dose at3 mg/kg IV (FIG. 24A), 3 mg/kg IP (FIG. 24B), or 10 mg/kg oral (PO)(FIG. 24C). Prior to dosing, mice were fasted overnight until 4 hourspost-dosing. Blood samples were collected at the indicated timepointsand analyzed by HPLC/MS/MS to evaluate the concentration of (14A). TheCmax after oral dosing was 255 ng/mL after 2 hours, the calculated T ½was approximately 5 hours, and the oral bioavailability was 44%. The IVclearance was 29/ml/min/kg, the Vss was 11.7 L/kg, and the IPbioavailability was 95%.

DEFINITIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure.Such compounds are useful, for example, as analytical tools or probes inbiological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic,” as used herein, refers to alkyl, alkenyl, alkynyl,and carbocyclic groups. Likewise, the term “heteroaliphatic” as usedherein, refers to heteroalkyl, heteroalkenyl, heteroalkynyl, andheterocyclic groups.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 10 carbon atoms(“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbonatoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl grouphas 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkylgroup has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, analkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments,an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In someembodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). Insome embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. Unless otherwise specified, each instance of an alkylgroup is independently unsubstituted (an “unsubstituted alkyl”) orsubstituted (a “substituted alkyl”) with one or more substituents. Incertain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl(e.g., —CH₃). In certain embodiments, the alkyl group is a substitutedC₁₋₁₀ alkyl.

As used herein, “haloalkyl” is a substituted alkyl group as definedherein wherein one or more of the hydrogen atoms are independentlyreplaced by a halogen, e.g., fluoro, bromo, chloro, or iodo.“Perhaloalkyl” is a subset of haloalkyl, and refers to an alkyl groupwherein all of the hydrogen atoms are independently replaced by ahalogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, thehaloalkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ haloalkyl”). In someembodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C₁₋₆haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 4 carbonatoms (“C₁₋₄ haloalkyl”). In some embodiments, the haloalkyl moiety has1 to 3 carbon atoms (“C₁₋₃ haloalkyl”). In some embodiments, thehaloalkyl moiety has 1 to 2 carbon atoms (“C₁₋₂ haloalkyl”). In someembodiments, all of the haloalkyl hydrogen atoms are replaced withfluoro to provide a perfluoroalkyl group. In some embodiments, all ofthe haloalkyl hydrogen atoms are replaced with chloro to provide a“perchloroalkyl” group. Examples of haloalkyl groups include —CF₃,—CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “heteroalkyl” refers to an alkyl group as defined hereinwhich further includes at least one heteroatom (e.g., 1, 2, 3, or 4heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e.,inserted between adjacent carbon atoms of) and/or placed at one or moreterminal position(s) of the parent chain. In certain embodiments, aheteroalkyl group refers to a saturated group having from 1 to 10 carbonatoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₁₀alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 9 carbon atoms and 1 or more heteroatoms within the parentchain (“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group isa saturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). Insome embodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms(“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has 2 to 7carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenylgroup has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, analkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In someembodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”).In some embodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”).The one or more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl.

As used herein, “heteroalkenyl” refers to an alkenyl group as definedherein which further includes at least one heteroatom (e.g., 1, 2, 3, or4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e.,inserted between adjacent carbon atoms of) and/or placed at one or moreterminal position(s) of the parent chain. In certain embodiments, aheteroalkenyl group refers to a group having from 2 to 10 carbon atoms,at least one double bond, and 1 or more heteroatoms within the parentchain (“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenylgroup has 2 to 9 carbon atoms at least one double bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₉ alkenyl”). In someembodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₈ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 7 carbon atoms, at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”).In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds)(“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 9carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynylgroup has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, analkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In someembodiments, an alkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”).In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms(“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has 2 carbonatoms (“C₂ alkynyl”). The one or more carbon-carbon triple bonds can beinternal (such as in 2-butynyl) or terminal (such as in 1-butynyl).Examples of C₂₋₄ alkynyl groups include, without limitation, ethynyl(C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄),and the like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and thelike. Additional examples of alkynyl include heptynyl (C₇), octynyl(C₈), and the like. Unless otherwise specified, each instance of analkynyl group is independently unsubstituted (an “unsubstitutedalkynyl”) or substituted (a “substituted alkynyl”) with one or moresubstituents. In certain embodiments, the alkynyl group is anunsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl groupis a substituted C₂₋₁₀ alkynyl.

As used herein, “heteroalkynyl” refers to an alkynyl group as definedherein which further includes at least one heteroatom (e.g., 1, 2, 3, or4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e.,inserted between adjacent carbon atoms of) and/or placed at one or moreterminal position(s) of the parent chain. In certain embodiments, aheteroalkynyl group refers to a group having from 2 to 10 carbon atoms,at least one triple bond, and 1 or more heteroatoms within the parentchain (“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynylgroup has 2 to 9 carbon atoms, at least one triple bond, and 1 or moreheteroatoms within the parent chain (“heteroC₂₋₉ alkynyl”). In someembodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₈ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 7 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 9 ring carbon atoms (“C₃₋₉ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C₃₋₈carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ringcarbon atoms (“C₃₋₇ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C₄₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ringcarbon atoms (“C₅₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 5 to 10 ring carbon atoms (“(C₅₋₁₀ carbocyclyl”). ExemplaryC₃₋₆ carbocyclyl groups include, without limitation, cyclopropyl (C₃),cyclopropenyl (C₃), cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl(C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆),cyclohexadienyl (C₆), and the like. Exemplary C₃₋₈ carbocyclyl groupsinclude, without limitation, the aforementioned C₃₋₆ carbocyclyl groupsas well as cycloheptyl (C₇), cycloheptenyl (C₇), cycloheptadienyl (C₇),cycloheptatrienyl (C₇), cyclooctyl (C₈), cyclooctenyl (C₈),bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl (C₈), and the like.Exemplary C₃₋₁₀ carbocyclyl groups include, without limitation, theaforementioned C₃₋₈ carbocyclyl groups as well as cyclononyl (C₉),cyclononenyl (C₉), cyclodecyl (C₁₀), cyclodecenyl (C₁₀),octahydro-1H-indenyl (C₉), decahydronaphthalenyl (C₁₀),spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 9 ringcarbon atoms (“C₃₋₉ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“(C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₀ cycloalkyl.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical ofa 3- to 14-membered non-aromatic ring system having ring carbon atomsand 1 to 4 ring heteroatoms, wherein each heteroatom is independentlyselected from nitrogen, oxygen, and sulfur (“3-14 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) ortricyclic system (“tricyclic heterocyclyl”)), and can be saturated orcan contain one or more carbon-carbon double or triple bonds.Heterocyclyl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heterocyclyl” also includes ring systems whereinthe heterocyclyl ring, as defined above, is fused with one or morecarbocyclyl groups wherein the point of attachment is either on thecarbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. Unless otherwise specified, each instance of heterocyclylis independently unsubstituted (an “unsubstituted heterocyclyl”) orsubstituted (a “substituted heterocyclyl”) with one or moresubstituents. In certain embodiments, the heterocyclyl group is anunsubstituted 3-14 membered heterocyclyl. In certain embodiments, theheterocyclyl group is a substituted 3-14 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyland pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary6-membered heterocyclyl groups containing 3 heteroatoms include, withoutlimitation, triazinanyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 □ electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by an aryl group, as defined herein, whereinthe point of attachment is on the alkyl moiety.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ringsystem (e.g., having 6, 10, or 14 □ electrons shared in a cyclic array)having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). Inheteroaryl groups that contain one or more nitrogen atoms, the point ofattachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by a heteroaryl group, as defined herein,wherein the point of attachment is on the alkyl moiety.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that doesnot contain a double or triple bond, i.e., the ring contains all singlebonds.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups, as defined herein, are, in certain embodiments,optionally substituted. Optionally substituted refers to a group whichmay be substituted or unsubstituted (e.g., “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The present invention contemplates any andall such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary substituents include, but are not limited to, halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂, —N(R^(bb))₂,—N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa), —SSR^(cc),—C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa), —OC(═O)R^(aa),—OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa),—NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa),—C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃,—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa),—P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂,—OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂,—P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂,—NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂,—OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl,C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂,—NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminalR^(dd) substituents can be joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₄₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

In certain embodiments, an exemplary substituent is selected from thegroup consisting of halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OR^(aa), —N(R^(bb))₂, —SH, —SR^(aa), —SSR^(cc), —C(═O)R^(aa), —CO₂H,—CHO, —CO₂R^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂,—OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa),—SO₂N(R^(bb))₂, —SO₂R^(aa), —S(═O)R^(aa), C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro,—F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

As used herein, a “leaving group” is an art-understood term referring toa molecular fragment that departs with a pair of electrons inheterolytic bond cleavage, wherein the molecular fragment is an anion orneutral molecule. See, for example, Smith, March Advanced OrganicChemistry 6th ed. (501-502). Exemplary leaving groups include, but arenot limited to, halo (e.g., chloro, bromo, iodo) and —OSO₂R^(aa),wherein R^(aa) as defined herein. The group —OSO₂R^(aa) encompassesleaving groups such as tosyl, mesyl, and besyl, wherein R^(aa) isoptionally substituted alkyl (e.g., —CH₃) or optionally substituted aryl(e.g., phenyl, tolyl).

As used herein, the term “hydroxyl” or “hydroxy” refers to the group—OH. The term “substituted hydroxyl” or “substituted hydroxyl,” byextension, refers to a hydroxyl group wherein the oxygen atom directlyattached to the parent molecule is substituted with a group other thanhydrogen, and includes groups selected from —OR^(aa), —ON(R^(bb))₂,—OC(═O)SR^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂,—OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂,—OS(═O)R^(aa), —OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃,—OP(═O)₂R^(aa), —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —OP(═O)₂N(R^(bb))₂,and —OP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein.

As used herein, the term “thiol” or “thio” refers to the group —SH. Theterm “substituted thiol” or “substituted thio,” by extension, refers toa thiol group wherein the sulfur atom directly attached to the parentmolecule is substituted with a group other than hydrogen, and includesgroups selected from —SR^(aa), —S═SR^(cc), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —SC(═O)OR^(aa), and —SC(═O)R^(aa), wherein R^(aa) andR^(cc) are as defined herein.

As used herein, the term, “amino” refers to the group —NH₂. The term“substituted amino,” by extension, refers to a monosubstituted amino ora disubstituted amino, as defined herein. In certain embodiments, the“substituted amino” is a monosubstituted amino or a disubstituted aminogroup.

As used herein, the term “monosubstituted amino” refers to an aminogroup wherein the nitrogen atom directly attached to the parent moleculeis substituted with one hydrogen and one group other than hydrogen, andincludes groups selected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

As used herein, the term “disubstituted amino” refers to an amino groupwherein the nitrogen atom directly attached to the parent molecule issubstituted with two groups other than hydrogen, and includes groupsselected from —N(R^(bb))₂, —NR^(bb) C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

As used herein, the term “sulfonyl” refers to a group selected from—SO₂N(R^(bb))₂, —SO₂R^(aa), and —SO₂OR^(aa), wherein R^(aa) and R^(bb)are as defined herein.

As used herein, the term “sulfinyl” refers to the group —S(═O)R^(aa),wherein R^(aa) is as defined herein.

As used herein, the term “carbonyl” refers a group wherein the carbondirectly attached to the parent molecule is sp² hybridized, and issubstituted with an oxygen, nitrogen or sulfur atom, e.g., a groupselected from ketones (—C(═O)R^(aa)), carboxylic acids (—CO₂H),aldehydes (—CHO), esters (—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)),amides (—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), andimines (—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)),—C(═NR^(bb))N(R^(bb))₂), wherein R^(aa) and R^(bb) are as definedherein.

As used herein, the term “silyl” refers to the group —Si(R^(aa))₃,wherein R^(aa) is as defined herein.

As used herein, the term “oxo” refers to the group ═O, and the term“thiooxo” refers to the group ═S.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substituents include, but are not limitedto, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to an N atom are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, andwherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined herein. Nitrogen protecting groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethyl silyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

Other Definitions

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptablesalts of the compounds of this invention include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example mammals [e.g., primates (e.g., cynomolgusmonkeys, rhesus monkeys); commercially relevant mammals such as cattle,pigs, horses, sheep, goats, cats, and/or dogs], birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), reptiles, amphibians, and fish. In certain embodiments, thenon-human animal is a mammal. The non-human animal may be a male orfemale and at any stage of development. A non-human animal may be atransgenic animal.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified disease, disorder or condition,which reduces the severity of the disease, disorder or condition, orretards or slows the progression of the disease, disorder or condition(“therapeutic treatment”), and also contemplates an action that occursbefore a subject begins to suffer from the specified disease, disorderor condition (“prophylactic treatment”).

In general, the “effective amount” of a compound refers to an amountsufficient to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a compound of the invention may vary depending on such factors as thedesired biological endpoint, the pharmacokinetics of the compound, thedisease being treated, the mode of administration, and the age, health,and condition of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment of a disease, disorder orcondition, or to delay or minimize one or more symptoms associated withthe disease, disorder or condition. A therapeutically effective amountof a compound means an amount of therapeutic agent, alone or incombination with other therapies, which provides a therapeutic benefitin the treatment of the disease, disorder or condition. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of disease orcondition, or enhances the therapeutic efficacy of another therapeuticagent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease, disorder or condition, or one or more symptoms associated withthe disease, disorder or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount of atherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease,disorder or condition. The term “prophylactically effective amount” canencompass an amount that improves overall prophylaxis or enhances theprophylactic efficacy of another prophylactic agent.

As used herein “modulating” refers to the ability of a compound toincrease or inhibit a particular biological process (e.g., kinaseactivity, overexpression), e.g., for example in a cell (e.g., in vitrosuch as a cell in a cell culture, or in vivo such as a cell in asubject) relative to vehicle.

As used herein “inhibition”, “inhibiting”, “inhibit” and “inhibitor”,and the like, refer to the ability of a compound to reduce, slow, haltor prevent activity of a particular biological process (e.g., kinaseactivity, overexpression), e.g., for example in a cell (e.g., in vitrosuch as a cell in a cell culture, or in vivo such as a cell in asubject) relative to vehicle.

As used herein “increasing” or “increase”, and the like, refer to theability of a compound to stimulate activity of a particular biologicalprocess (e.g., kinase activity), e.g., for example in a cell (e.g., invitro such as a cell in a cell culture, or in vivo such as a cell in asubject) relative to vehicle.

Detailed Description of Certain Embodiments of the Invention

As generally described herein, provided are new cortistatin analogswhich may be synthesized, in part, by reductive amination of a ketone ofFormula (B) to provide an aminated product of Formula (A), optionallyvia an imine intermediate of Formula (C). Further provided are newcortistatin analogs of Formulae (D) and (E). See, e.g., Scheme 1, supra.

and pharmaceutically acceptable salts, quaternary amine salts, orN-oxides thereof, wherein:

R¹ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, —OR^(A), —SR^(A), —N(R^(A))₂,—C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂, —S(═O)₂R^(A), or a nitrogenprotecting group;

R² is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A),—C(═O)N(R^(A))₂, —S(═O)₂R^(A), or a nitrogen protecting group;

or R¹ and R² are joined to form an optionally substituted heterocyclylor optionally substituted heteroaryl;

R³ is hydrogen or optionally substituted alkyl;

R⁴ is hydrogen, halogen, optionally substituted alkyl, or —Si(R^(A))₃;

R⁵ is hydrogen, halogen, or optionally substituted alkyl;

each instance of

, designated as (a), (b), and (c), represents a single or double bond,provided that when

designated as (c) represents a double bond, then one of R^(B1) andR^(B2) is absent, and provided that when

designated as (c) represents a single bond, then both R^(B1) and R^(B2)are present;

each instance of R^(B1) and R^(B2) is, independently, hydrogen,-L₁-R^(B3), or —X^(A)R^(A) wherein X^(A) is —O—, —S—, or —N(R^(A))—; orR^(B1) and R^(B2) are joined to form an oxo group, provided that atleast one of R^(B1) and R^(B2) is not hydrogen;

L¹ is a bond, —C(═O)—, —C(═O)O—, —C(═O)S—, —C(═O)N(R^(L))—, or—N(R^(L))—(C(R^(LL))₂)_(p)—, wherein R^(L) is hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group, each instance ofR^(LL) is independently hydrogen, halogen, or optionally substitutedalkyl, and p is 0, 1, or 2;

R^(B3) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl, provided that when L₁ is abond, then R^(B3) is not hydrogen;

each instance of R^(A) is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,carbonyl, silyl, an oxygen protecting group when attached to oxygen, asulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen, optionally when attached toN the two R^(A) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring, and optionallywhen R^(B1) and R^(B2) are each —X^(A)R^(A) then two R^(A) groups may bejoined to form an optionally substituted heterocyclyl ring; and

R^(O) is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A),—C(═O)N(R^(A))₂, or an oxygen protecting group.

It is generally understood that any atom encompassed by any of theformula described herein may be replaced with an isotope of that atom,e.g., for example, a hydrogen atom (¹H) may be replaced with a deuterium(²H, D) or tritium (³H, T) atom, a carbon atom (¹²C) may be replacedwith its ¹⁴C isotope, and a fluorine atom (¹⁸F) may be replaced by its¹⁹F isotope.

In general, reductive amination of Formula (B) generally provides bothalpha and beta aminated isomers encompassed by Formula (A), referred toherein as Formula (A-1), the beta isomer, and Formula (A-2), the alphaisomer, and the beta isomer is typically the major product of thereaction. The alpha isomer shares C3 stereochemistry with othercortistatin natural products. See, e.g., FIG. 1. Furthermore, reductionof the ketone generally provides both alpha and beta reduced isomersencompassed by Formula (D), referred to herein as Formula (D-1), thebeta isomer, and Formula (D-2), the alpha isomer. Subsequent protectionof these isomers of Formula (D) respectively provides Formula (E-1), thebeta isomer, and Formula (E-2), the alpha isomer.

The (2.4 Å) crystal structure of a cortistatin A/CDK8/cyclin C ternarycomplex (see, e.g., FIG. 16) reveals that cortistatin A exhibitsremarkable shape complementarity with the ATP-binding pocket of CDK8 andthere is an apparent cation-π interaction between the chargedN,N-dimethylammonium ion of cortistatin A and Trp105 of CDK8, which arewithin close contact (3.4 Å). CDK8 and CDK19 are the only CDKs with Trpat amino acid 105, suggesting that the cation-π interaction as well ashydrophobic contacts between cortistatin A and Trp105 might be importantfor the high affinity and selectivity of cortistatin A for CDK8. Basedon this crystal structure, the beta isomer was predicted to stericallyclash with Trp105.

The crystal structure also revealed that the A ring hydroxyl groups oncortistatin A might be dispensible for binding, as they do not formhydrogen bonds with CDK8 and orient away from CDK8, outside of thecortistatin A binding pocket. In addition, cortistatin J, which lacksthe hydroxyl groups present in cortistatin A, is only 8-fold less potentat inhibiting HUVEC proliferation (see, e.g., Aoki, et al., Bioorg. Med.Chem. (2007) 15:6758-6762) and certain synthetic cortistatin A analogswith replacement of both the amine and hydroxyl substituents retainedpotent HUVEC antiproliferative activity (see, e.g., Nicolaou, et al., J.Am. Chem. Soc. (2009) 131: 10587-10597).

However, and most surprisingly, beta isomers of Formula (A), which donot contain any hydroxyl groups on the A ring, have been found to beequipotent, or more potent, than cortistatin A at inhibiting CDK8 kinaseactivity and the proliferation of AML cells, and it has also been foundthat the corresponding alpha isomers, also which do not contain anyhydroxyl groups on the A ring, are also very potent.

It has also been discovered that compounds of Formula (B) have beenfound active against the growth of AML cell lines in culture and CDK8kinase activity in cells. It is envisioned that the imine of Formula(C), such as an oxime wherein R₁ is —OR^(A), will also be active.

Furthermore, despite the loss of the charged N,N-dimethylammonium ioncation-pi interaction, the compounds of Formula (D) and (E) are alsosurprisingly highly active.

Quaternary Amine Salts and N-Oxides

In certain embodiments, as provided herein, a compound of Formula (A),(B), (C), (D), or (E) may comprise a quaternary amine salt and/or anN-oxide.

A “quaternary amine salt” as used herein refers to an amino groupwherein the nitrogen atom comprises four valence bonds (e.g., issubstituted with four groups which may be hydrogen and/or non-hydrogengroups) such that the nitrogen atom is positively charged and the chargeis balanced (neutralized) with a counteranion (e.g., X^(C) as definedherein).

An “N-oxide” as used herein refers to an amino group wherein thenitrogen atom comprises four valence bonds (e.g, is substituted withfour groups which may be hydrogen and/or non-hydrogen groups, whereinone group directly attached to the nitrogen atom is an oxidyl group(—O^(⊖))) such that the nitrogen atom is positively charged, and whereinthe oxidyl group balances (neutralizes) the positive charge of thenitrogen atom.

It should be understood that any one of Formula (A), (B), (C), (D), or(E) may comprise quaternary amine salt and/or N-oxide groups at anyposition where an amino group may be located.

In particular, compounds of Formula (A) may comprise a quaternary aminesalt or N-oxide group at the C₃ position (also referred to as a“quaternary C3-amine salt” and “C3-N-oxide”), which comprises the aminogroup —NR₁R₂ attached to Ring A.

In certain embodiments, the amino group

at the C₃ position may be an quaternary amine salt formula

e.g., to provide a compound of Formula (A-QA):

wherein

, R¹, R², R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

Y is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, oroptionally substituted heteroaryl; and

X^(C) is a counteranion.

A quaternary C3-amine salt may be formed by reaction of the freeC3-amine with a group Y—X^(C), wherein Y is defined above (e.g.,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, or optionallysubstituted heterocyclyl), and X^(C) is a leaving group as definedherein. The counterion X^(C) resulting therefrom may be exchanged withanother counterion X^(C) by ion exchange methods, e.g., ion exchangechromatography. Exemplary X^(C) counterions include but are not limitedto halide ions (e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻,HSO₄ ⁻, sulfonate ions (e.g., methansulfonate,trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate,10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonicacid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), andcarboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate,glycerate, lactate, tartrate, glycolate, and the like). In certainembodiments, Y is optionally substituted alkyl (e.g., methyl). Incertain embodiments, X^(C) is a halide ion.

In certain embodiments, the quaternary amine salt of Formula (A-QA) isthe beta (A-1-QA) or alpha (A-2-QA) isomer of the following Formula:

wherein

, R¹, R², R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein.

Alternatively, in certain embodiments, the amino group

at the C₃ position may be an N-oxide of formula

e.g., to provide a compound of Formula (A-NO):

wherein

, R¹, R², R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein.

In certain embodiments, the N-oxide of Formula (A-NO) is the beta(A-1-NO) or alpha (A-2-NO) isomer of the following Formula:

wherein

, R¹, R², R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein.

It has been discovered that C₃ N-oxide of cortistatin A is equipotentwith cortistatin A in inhibition of HUVEC proliferation. See, e.g.,FIGS. 17A and 17B.

It has further been discovered that beta N-oxides of Formula (A-1-NO),as described herein, have increased stability in liver microsomescompared to the corresponding free beta amino analog of Formula (A-1).See, e.g., FIG. 19, demonstrating increased stability of 14B—N-oxidecompared to compound 14B.

Groups R¹ and R²

As generally defined herein, in certain embodiments of Formula (A) and(C), R¹ is hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, —OR^(A), —SR^(A),—N(R^(A))₂, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂, —S(═O)₂R^(A), ora nitrogen protecting group.

Furthermore, in certain embodiments of Formula (A), R² is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂,—S(═O)₂R^(A), or a nitrogen protecting group.

For example, in certain embodiments of Formula (A), at least one of R¹and R² is hydrogen. In certain embodiments of Formula (A), both of R¹and R² is hydrogen. In certain embodiments of Formula (A), one of R¹ andR² is hydrogen and the other is a non-hydrogen group, e.g, optionallysubstituted alkyl. In certain embodiments of Formula (C), R¹ ishydrogen.

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted alkyl, e.g., optionally substituted C₁₋₆alkyl. Incertain embodiments of Formula (A), each instance of R¹ and R² isindependently optionally substituted alkyl. In certain embodiments ofFormula (C), R¹ is optionally substituted alkyl, e.g., optionallysubstituted C₁₋₆alkyl. In certain embodiments, R¹ and/or R² isoptionally substituted C₁alkyl, optionally substituted C₂alkyl,optionally substituted C₃alkyl, optionally substituted C₄alkyl,optionally substituted C₅alkyl, or optionally substituted C₆alkyl. Incertain embodiments, R¹ and/or R² is optionally substituted methyl (C₁),optionally substituted ethyl (C₂), optionally substituted n-propyl (C₃),optionally substituted isopropyl (C₃), optionally substituted n-butyl(C₄), or optionally substituted t-butyl (C₄). In certain embodiments, R¹and/or R² is alkyl substituted with one or more halogen substitutents(e.g., fluoro). In certain embodiments, R¹ and/or R² is —CH₃ or —CF₃. Incertain embodiments, each instance of R¹ and R² is independently —CH₃ or—CF₃. In certain embodiments, R¹ and/or R² is alkyl substituted with oneor more halogen (e.g., fluoro), amino (—NH₂), substituted amino,hydroxyl (—OH), substituted hydroxyl, thiol (—SH), substituted thiol, orsulfonyl substituents. In certain embodiments, R¹ and/or R² is alkylsubstituted with an optionally substituted carbocyclyl (e.g.,cyclopropyl) or optionally substituted heterocyclyl (e.g., oxetanyl)ring.

For example, in certain embodiments, at least one of R¹ and R² is agroup of formula:

e.g., to provide a compound of Formula (A-f), (A-1-f) or (A-2-f):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof,wherein

, R¹, R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

p is 1, 2, 3, 4, 5, or 6; and

Z is —CH₂X^(Z), —CH(X^(Z))₂, —C(X^(Z))₃, —OR^(Z), —SR^(Z), —N(R^(Z))₂,—S(O)₂N(R^(Z))₂,

wherein each instance of R^(Z) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(Z), —C(═O)OR^(Z), —C(═O)N(R^(Z))₂, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(Z) groupsmay be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring;

each instance of X^(Z) is independently fluoro, chloro, bromo, or iodo;and

w is an integer between 1 and 10, inclusive.

In certain embodiments, both instances of R¹ and R² are independently agroup of formula

In certain embodiments, p is 1. In certain embodiments, p is 2. Incertain embodiments, p is 3. In certain embodiments, w is 1, 2, 3, or 4.In certain embodiments, R^(Z) is hydrogen or optionally substitutedalkyl (e.g., —CH₃). In certain embodiments, Z is —OR^(Z), e.g., —OH or—OR^(Z) wherein R^(Z) is a non-hydrogen group, e.g., wherein R^(Z) isoptionally substituted alkyl such as —CH₃. In certain embodiments, Z is—N(R^(Z))₂, e.g., —NH₂, —NHR^(Z), or —N(R^(Z))₂ wherein R^(Z) is anon-hydrogen group, e.g., wherein R^(Z) is optionally substituted alkylsuch as —CH₃. In certain embodiments, Z is —CH₂X^(Z), —CH(X^(Z))₂,—C(X^(Z))₃, e.g., wherein X^(Z) is fluoro. In certain embodiments, Z is—S(O)₂N(R^(Z))₂, e.g., —S(O)₂NH₂ or —S(O)₂NHCH₃.

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted alkenyl, e.g., optionally substitutedC₂₋₆alkenyl. In certain embodiments of Formula (A), both of R¹ and R² isoptionally substituted alkenyl. In certain embodiments of Formula (C),R¹ is optionally substituted alkenyl, e.g., optionally substitutedC₂₋₆alkenyl. In certain embodiments, R¹ and/or R² is optionallysubstituted C₂alkenyl, optionally substituted C₃alkenyl, optionallysubstituted C₄alkenyl, optionally substituted C₅alkenyl, or optionallysubstituted C₆alkenyl. In certain embodiments, R¹ and/or R² isoptionally substituted vinyl (C₂) or optionally substituted allyl (C₃).

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted alkynyl, e.g., optionally substitutedC₂₋₆alkynyl. In certain embodiments of Formula (A), both of R¹ and R² isoptionally substituted alkynyl. In certain embodiments of Formula (C),R¹ is optionally substituted alkynyl, e.g., optionally substitutedC₂₋₆alkynyl. In certain embodiments, R¹ and/or R² is optionallysubstituted C₂alkynyl, optionally substituted C₃alkynyl, optionallysubstituted C₄alkynyl, optionally substituted C₅alkynyl, or optionallysubstituted C₆alkynyl. In certain embodiments, R¹ and/or R² isoptionally substituted acetylenyl (C₂) or optionally substitutedpropargyl (C₃).

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted carbocyclyl, e.g., optionally substitutedC₃₋₆carbocyclyl. In certain embodiments of Formula (A), both of R¹ andR² is optionally substituted carbocyclyl. In certain embodiments ofFormula (C), R¹ is optionally substituted carbocyclyl, e.g., optionallysubstituted C₃-6carbocyclyl. In certain embodiments, R¹ and/or R² isoptionally substituted C₃ carbocyclyl, optionally substituted C₄carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl. In certain embodiments, R¹ and/or R² isoptionally substituted cyclopropyl (C₃), optionally substitutedcyclobutyl (C₄), optionally substituted cyclopenyl (C₅), or optionallysubstituted cyclohexyl (C₆).

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted heterocyclyl, e.g., optionally substituted 3-6membered heterocyclyl. In certain embodiments of Formula (A), both of R¹and R² is optionally substituted heterocyclyl. In certain embodiments ofFormula (C), R¹ is optionally substituted heterocyclyl, e.g., optionallysubstituted 3-6 membered heterocyclyl. In certain embodiments, R¹ and/orR² is optionally substituted 3-membered heterocyclyl (e.g., optionallysubstituted oxetanyl), optionally substituted 4-membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl, e.g., optionally substituted6-membered heterocyclyl comprising 1 or 2 heteroatoms selected fromoxygen, sulfur, or nitrogen.

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted aryl, e.g., optionally substituted phenyl. Incertain embodiments of Formula (A), both of R¹ and R² is optionallysubstituted phenyl. In certain embodiments of Formula (C), R¹ isoptionally substituted aryl, e.g., optionally substituted phenyl.

In certain embodiments of Formula (A), at least one of R¹ and R² isoptionally substituted heteroaryl, e.g., optionally substituted 5-6membered heteroaryl. In certain embodiments of Formula (A), both of R¹and R² is optionally substituted heteroaryl. In certain embodiments ofFormula (C), R¹ is optionally substituted heteroaryl, e.g., optionallysubstituted 5-6 membered heteroaryl.

In certain embodiments of Formula (A) and (C), R¹ is —OR^(A), e.g., —OHor —OCH₃. In certain embodiments of Formula (A) and (C), R¹ is —SR^(A).In certain embodiments of Formula (A) and (C), R¹ is —N(R^(A))₂.

In certain embodiments of Formula (A), at least one of R¹ and R² is—C(═O)R^(A), —C(═O)OR^(A), or —C(═O)N(R^(A))₂. In certain embodiments ofFormula (C), R¹ is —C(═O)R^(A), —C(═O)OR^(A), or —C(═O)N(R^(A))₂.

In certain embodiments of Formula (A), at least one of R¹ and R² is anitrogen protecting group. In certain embodiments of Formula (C), R¹ isa nitrogen protecting group.

Furthermore, as generally defined herein, in certain embodiments ofFormula (A), R¹ and R² are joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl.

In certain embodiments of Formula (A), at least one of R¹ and R² is—S(O)₂R^(A). In certain embodiments, R^(A) optionally substituted alkyl(e.g., —CH₃). In certain embodiments of Formula (A), one of R¹ and R² is—S(O)₂R^(A) and the other is optionally substituted alkyl (e.g., —CH₃).In certain embodiments of Formula (C), R¹ is —S(O)₂R^(A).

In certain embodiments of Formula (A), R¹ and R² are joined to form anoptionally substituted heterocyclyl, e.g., an optionally substituted 3-6membered heterocyclyl. In certain embodiments of Formula (A), R¹ and R²are joined to form an optionally substituted 3-membered heterocyclyl, anoptionally substituted 4-membered heterocyclyl, optionally substituted5-membered heterocyclyl, or an optionally substituted 6-memberedheterocyclyl. In certain embodiments, R¹ and R² are joined to form anoptionally substituted 3-membered heterocyclyl, i.e., an optionallysubstituted aziridinyl. In certain embodiments, R¹ and R² are joined toform an optionally substituted 4-membered heterocyclyl, e.g., anoptionally substituted azetidinyl. In certain embodiments, R¹ and R² arejoined to form an optionally substituted 5-membered heterocyclyl, e.g.,an optionally substituted pyrrolidinyl or optionally substitutedimidazolidine-2,4-dione. In certain embodiments, R¹ and R² are joined toform an optionally substituted 6-membered heterocyclyl, e.g., anoptionally substituted piperidinyl, optionally substitutedtetrahydropyranyl, optionally substituted dihydropyridinyl, optionallysubstituted thianyl, optionally substituted piperazinyl, optionallysubstituted morpholinyl, optionally substituted dithianyl, optionallysubstituted dioxanyl, or optionally substituted triazinanyl.

For example, in certain embodiments, R¹ and R² are joined to form agroup of formula:

e.g., to provide a compound of Formula (A-a), (A-1-a) or (A-2-a):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, wherein

, R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

G is —O—, —S—, —NH—, —NR⁷—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—;

each instance of R⁷ is independently halogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, amino,substituted amino, hydroxyl, substituted hydroxyl, thiol, substitutedthiol, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group whenattached to a nitrogen atom;

optionally wherein two R⁷ groups are joined to form an optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, an optionally substituted heteroaryl ring, or an oxo(═O) group; and

n is 0, 1, 2, 3, or 4.

In certain embodiments, R¹ and R² are joined to form a group of formula:

e.g., to provide a compound of Formula (A-b), (A-1-b), or (A-2-b):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, wherein

, R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

G is —O—, —S—, —NH—, —NR⁷—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—;

each instance of R⁷ is independently halogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, amino,substituted amino, hydroxyl, substituted hydroxyl, thiol, substitutedthiol, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group whenattached to a nitrogen atom;

optionally wherein two R⁷ groups are joined to form an optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, an optionally substituted heteroaryl ring, or an oxo(═O) group; and

n is 0, 1, 2, 3, or 4.

In certain embodiments, R¹ and R² are joined to form a group of formula:

e.g., to provide a compound of Formula (A-c), (A-1-c), (A-2-c):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, wherein

, R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

G is —O—, —S—, —NH—, —NR⁷—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—;

each instance of R⁷ is independently halogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, amino,substituted amino, hydroxyl, substituted hydroxyl, thiol, substitutedthiol, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group whenattached to a nitrogen atom;

optionally wherein two R⁷ groups are joined to form an optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, an optionally substituted heteroaryl ring, or an oxo(═O) group; and

n is 0, 1, 2, 3, or 4.

In certain embodiments, n is 0, and the ring system formed by thejoining of R¹ and R² is not substituted with an R⁷ group as definedherein. In certain embodiments, n is 1, 2, 3, or 4, and the ring systemis substituted with 1, 2, 3, or 4 R⁷ groups as defined herein. Incertain embodiments, n is 1. In certain embodiments, n is 2. In certainembodiments, n is 3. In certain embodiments, n is 4.

In certain embodiments, wherein n is not 0 (i.e., n is 1, 2, 3, or 4)and at least one R⁷ is attached to a carbon atom, the R⁷ is halogen(e.g., fluoro), hydroxyl, substituted hydroxyl, or carbonyl (e.g.,—CO₂H). In certain embodiments, wherein n is not 0 (i.e., n is 1, 2, 3,or 4) and two R⁷ groups are attached to the same carbon atom, the two R⁷groups are each halogen, e.g., fluoro. In certain embodiments, wherein nis not 0 (i.e., n is 1, 2, 3, or 4) and two R⁷ groups are attached tothe same carbon atom, the two R⁷ groups are joined to form an optionallysubstituted carbocyclyl ring or optionally substituted heterocyclyl ring(e.g., optionally substituted oxetanyl ring). In certain embodiments,wherein n is not 0 (i.e., n is 1, 2, 3, or 4) and two R⁷ groups areattached to a different carbon atom, the two R⁷ groups are joined toform an optionally substituted carbocyclyl ring or optionallysubstituted heterocyclyl ring.

In certain embodiments, G is —O—. In certain embodiments, G is —NR⁷—,e.g., wherein R⁷ is optionally substituted alkyl (e.g., —CH₃). Incertain embodiments, G is —CH(R⁷)— or —C(R⁷)₂— wherein at least one R⁷is hydroxyl, substituted hydroxyl, or carbonyl (e.g., —CO₂H).

In certain embodiments, the group

In certain embodiments, the group

In certain embodiments, the group

In certain embodiments of Formula (A), R¹ and R² are joined to form anoptionally substituted heteroaryl, e.g., an optionally substituted5-membered heteroaryl or optionally substituted 6-membered heteroaryl.

Group R^(O)

As generally defined herein, for Formula (E), R^(O) is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂, oran oxygen protecting group.

In certain embodiments of Formula (E), R^(O) is optionally substitutedalkyl, e.g., optionally substituted C₁₋₆alkyl, e.g., optionallysubstituted C₁alkyl, optionally substituted C₂alkyl, optionallysubstituted C₃alkyl, optionally substituted C₄alkyl, optionallysubstituted C₅alkyl, or optionally substituted C₆alkyl. In certainembodiments, R^(O) is optionally substituted methyl (C₁), optionallysubstituted ethyl (C₂), optionally substituted n-propyl (C₃), optionallysubstituted isopropyl (C₃), optionally substituted n-butyl (C₄), oroptionally substituted t-butyl (C₄). In certain embodiments, R^(O) isalkyl substituted with one or more halogen substitutents (e.g., fluoro).In certain embodiments, R^(O) is —CH₃ or —CF₃. In certain embodiments,R^(O) is alkyl substituted with one or more halogen (e.g., fluoro),amino (—NH₂), substituted amino, hydroxyl (—OH), substituted hydroxyl,thiol (—SH), substituted thiol, or sulfonyl substituents. In certainembodiments, R^(O) is alkyl substituted with an optionally substitutedcarbocyclyl (e.g., cyclopropyl) or optionally substituted heterocyclyl(e.g., oxetanyl) ring.

For example, in certain embodiments, R^(O) is a group of formula:

e.g., to provide a compound of Formula (E-f), (E-1-f) or (E-2-f):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, wherein

R³, R⁴, R⁵, R^(B1), and R^(B2) are as defined herein; andwherein:

p is 1, 2, 3, 4, 5, or 6; and

Z is —CH₂X^(Z), —CH(X^(Z))₂, —C(X^(Z))₃, —OR^(Z), —SR^(Z), —N(R^(Z))₂,—S(O)₂N(R^(Z))₂,

wherein each instance of R^(Z) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(Z), —C(═O)OR^(Z), —C(═O)N(R^(Z))₂, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(Z) groupsmay be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring;

each instance of X^(Z) is independently fluoro, chloro, bromo, or iodo;and

w is an integer between 1 and 10, inclusive.

In certain embodiments, p is 1. In certain embodiments, p is 2. Incertain embodiments, p is 3. In certain embodiments, w is 1, 2, 3, or 4.In certain embodiments, R^(Z) is hydrogen or optionally substitutedalkyl (e.g., —CH₃). In certain embodiments, Z is —OR^(Z), e.g., —OH or—OR^(Z) wherein R^(Z) is a non-hydrogen group, e.g., wherein R^(Z) isoptionally substituted alkyl such as —CH₃. In certain embodiments, Z is—N(R^(Z))₂, e.g., —NH₂, —NHR^(Z), or —N(R^(Z))₂ wherein R^(Z) is anon-hydrogen group, e.g., wherein R^(Z) is optionally substituted alkylsuch as —CH₃. In certain embodiments, Z is —CH₂X^(Z), —CH(X^(Z))₂,—C(X^(Z))₃, e.g., wherein X^(Z) is fluoro. In certain embodiments, Z is—S(O)₂N(R^(Z))₂, e.g., —S(O)₂NH₂ or —S(O)₂NHCH₃.

In certain embodiments of Formula (E), R^(O) is optionally substitutedalkenyl, e.g., optionally substituted C₃₋₆alkenyl, e.g., optionallysubstituted C₃alkenyl, optionally substituted C₄alkenyl, optionallysubstituted C₅alkenyl, or optionally substituted C₆alkenyl.

In certain embodiments of Formula (E), R^(O) is optionally substitutedalkynyl, e.g., optionally substituted C₃₋₆alkynyl, e.g., optionallysubstituted C₃alkynyl, optionally substituted C₄alkynyl, optionallysubstituted C₅alkynyl, or optionally substituted C₆alkynyl.

In certain embodiments of Formula (E), R^(O) is optionally substitutedcarbocyclyl, e.g., optionally substituted C₃₋₆carbocyclyl, e.g.,optionally substituted C₃ carbocyclyl, optionally substituted C₄carbocyclyl, optionally substituted (C₅) carbocyclyl, or optionallysubstituted C₆ carbocyclyl. In certain embodiments, R^(O) is optionallysubstituted cyclopropyl (C₃), optionally substituted cyclobutyl (C₄),optionally substituted cyclopenyl (C₅), or optionally substitutedcyclohexyl (C₆).

In certain embodiments of Formula (E), R^(O) is optionally substitutedheterocyclyl, e.g., optionally substituted 3-6 membered heterocyclyl,e.g., optionally substituted 3-membered heterocyclyl (e.g., optionallysubstituted oxetanyl), optionally substituted 4-membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl, e.g., optionally substituted6-membered heterocyclyl comprising 1 or 2 heteroatoms selected fromoxygen, sulfur, or nitrogen.

In certain embodiments of Formula (E), at least one of R¹ and R² isoptionally substituted aryl, e.g., optionally substituted phenyl.

In certain embodiments of Formula (A), R^(O) is optionally substitutedheteroaryl, e.g., optionally substituted 5-6 membered heteroaryl.

In certain embodiments of Formula (E), R^(O) is —C(═O)R^(A),—C(═O)OR^(A), or —C(═O)N(R^(A))₂. In certain embodiments, R^(A) ishydrogen or optionally substituted alkyl (e.g., —CH₃). For example, incertain embodiments, R^(O) is —C(═O)CH₃, —C(═O)OCH₃, —C(═O)N(CH₃)₂, or—C(═O)NHCH₃.

In certain embodiments of Formula (E), R^(O) is an oxygen protectinggroup.

Group R³, R⁴, R⁵, and bonds (a), (b), and (c) of formula

As generally defined herein, for Formula (A), (B), (C), (D), and (E), R³is hydrogen or optionally substituted alkyl.

In certain embodiments, R³ is hydrogen. Such compounds are possibleusing starting materials such as 18-nor estrone.

In certain embodiments, R³ is optionally substituted alkyl, e.g., methyl(—CH₃). Such compounds are possible by using starting materials such asestrone and tetralone for methyl.

As generally defined herein, for Formula (A), (B), (C), (D), and (E), R⁴is hydrogen, halogen, optionally substituted alkyl, or —Si(R^(A))₃.

For example, in certain embodiments, R⁴ is hydrogen. In certainembodiments, R⁴ is optionally substituted alkyl, e.g., methyl. Incertain embodiments, R⁴ is —Si(R^(A))₃, e.g., wherein each instance ofR^(A) is independently optionally substituted alkyl or optionallysubstituted phenyl.

As generally defined herein, R⁵ is hydrogen, halogen, or optionallysubstituted alkyl. In certain embodiments, R⁵ is hydrogen. In certainembodiments, R⁵ is halogen (e.g., bromo, iodo, chloro). In certainembodiments, R⁵ is optionally substituted alkyl, e.g., methyl.

As generally defined herein, each instance of

, designated as (a), (b), and (c), represents a single or double bond,provided that when

designated as (c) represents a double bond, then one of R^(B1) andR^(B2) is absent, and provided that when

designated as (c) represents a single bond, then both R^(B1) and R^(B2)are present.

In certain embodiments, the bond

designated as (a) is a single bond. In certain embodiments, the bond

designated as (a) is a double bond.

In certain embodiments, the bond

designated as (b) is a single bond. In certain embodiments, the bond

designated as (b) is a double bond.

In certain embodiments, the bond

designated as (c) is a single bond. In certain embodiments, the bond

designated as (c) is a double bond, and R^(B2) is absent.

In certain embodiments, the bond

designated as (a) is double bond, and the bond

designated as (b) is a double bond. In this instance, in certainembodiments, the bond

designated as (c) is a double bond, and R^(B2) is absent. However, inthis instance, in other embodiments, the bond

designated as (c) is a single bond.

For example, in certain embodiments of Formula (A), (B), (C), (D), and(E), wherein R³ is methyl, R⁴ is hydrogen, R⁵ is hydrogen, and the bonddesignated (c) is a single bond, provided are compounds of Formula(A-d), (A-1-d), (A-2-d), (B-d), (C-d), (D-d), (D-1-d), (D-2-d), (E-d),(E-1-d), and (E-2-d):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R¹, R², R^(O), R^(B1), and R^(B2) are as definedherein.

In other embodiments of Formula (A), (B), (C), (D), and (E), wherein R³is methyl, R⁴ is hydrogen, the bond designated (c) is a double bond, andR^(B2) is absent, provided are compounds of Formula (A-e), (A-1-e),(A-2-e), (B-e), (C-e), (D-e), (D-1-e), (D-2-e), (E-e), (E-1-e), (E-2-e):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R¹, R², R^(O), R⁵, and R^(B1), are as defined herein.Groups R^(B1) and R^(B2)

As generally defined herein, each instance of R^(B1) and R^(B2) is,independently, hydrogen, -L₁-R^(B3), or —X^(A)R^(A) wherein X^(A) is—O—, —S—, or —N(R^(A))—, provided that at least one of R^(B1) and R^(B2)is not hydrogen; or R^(B1) and R^(B2) are joined to form an oxo group;

L₁ is a bond, —C(═O)—, —C(═O)O—, —C(═O)S—, —C(═O)N(R^(L))—, or—N(R^(L))—(C(R^(LL))₂)_(p)—, wherein R^(L) is hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group, each instance ofR^(LL) is independently hydrogen, halogen, or optionally substitutedalkyl, and p is 0, 1, or 2;

R^(B3) is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl, provided that when L₁ is abond, then R^(B3) is not hydrogen;

each instance of R^(A) is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,carbonyl, silyl, an oxygen protecting group when attached to oxygen, asulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen; optionally when attached toN the two R^(A) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring; and optionallywhen R^(B1) and R^(B2) are each —X^(A)R^(A) then two R^(A) groups may bejoined to form an optionally substituted heterocyclyl ring.

In certain embodiments, at least one instance of R^(B1) and R^(B2) is-L₁-R^(B3). In this instance, in certain embodiments, the other ofR^(B1) and R^(B2) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)). Forexample, in certain embodiments, when

designated as (c) represents a single bond, then R^(B1) is -L₁-R^(B3)and R^(B2) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)). In otherembodiments, when

designated as (c) represents a single bond, then R^(B2) is -L₁-R^(B3)and R^(B1) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)). Alternatively, incertain embodiments, when

designated as (c) represents a double bond, then R^(B1) is -L₁-R^(B3)and R^(B2) is absent.

For example, in certain embodiments of Formula (A), (B), (C), (D), and(E), wherein R^(B1) is -L₁-R^(B3), provided are compounds of Formula(A-g), (A-1-g), (A-2-g), (B-g), (C-g), (D-g), (D-1-g), (D-2-g), (E-g),(E-1-g), (E-2-g):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R^(O), R³, R⁴, R⁵, R^(B2), L₁, and R^(B3) are as definedherein.

In certain embodiments, L₁ is a bond, and R^(B3) is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, L₁ is —C(═O)—, —C(═O)O—, —C(═O)S—,—C(═O)N(R^(L))—, or —N(R^(L))—(C(R^(LL))₂)_(p)—, wherein R^(L) ishydrogen, optionally substituted alkyl, or a nitrogen protecting group,each instance of R^(LL) is independently hydrogen, halogen, oroptionally substituted alkyl, and R^(B3) is hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In certain embodiments, L₁ is —C(═O)—. Incertain embodiments, L₁ is —C(═O)O—. In certain embodiments, L₁ is—C(═O)S—. In certain embodiments, L₁ is —C(═O)N(R^(L))—. In certainembodiments, L₁ is —N(R^(L))—C(R^(LL))₂—. In certain embodiments, R^(L)is hydrogen or optionally substituted alkyl, e.g., methyl. In certainembodiments, each instance of R^(LL) is independently hydrogen,optionally substituted alkyl, e.g., methyl, or fluoro. In certainembodiments, p is 0. In certain embodiments, p is 1. In certainembodiments, p is 2.

In certain embodiments, when L₁ is —C(═O)—, —C(═O)O—, —C(═O)S—,—C(═O)N(R^(L))—, or —N(R^(L))—(C(R^(LL))₂)_(p)—, and R^(B3) is hydrogen,provided is a group of formula —C(═O)H, —C(═O)OH, —C(═O)SH,—C(═O)N(R^(L))H, or —N(R^(L))H.

However, in certain embodiments when L₁ is a bond or —C(═O)—, —C(═O)O—,—C(═O)S—, —C(═O)N(R^(L))—, or —N(R^(L))—(C(R^(LL))₂)_(p)—, R^(B3) isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, R^(B3) is an acyclic group, e.g., R^(B3) is anoptionally substituted alkyl, optionally substituted alkenyl, oroptionally substituted alkynyl. In certain embodiments, R^(B3) isoptionally substituted alkyl, e.g., optionally substituted C₁₋₆alkyl. Incertain embodiments, R^(B3) is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆alkenyl. In certain embodiments, R^(B3) isoptionally substituted alkynyl, e.g., optionally substitutedC₂₋₆alkynyl.

However, in certain embodiments, R^(B3) is a cyclic group, e.g., R^(B3)is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl. In certain embodiments, R^(B3) is a nonaromatic cyclicgroup, e.g., in certain embodiments, R^(B3) is optionally substitutedcarbocyclyl or optionally substituted heterocyclyl. In certainembodiments, R^(B3) is an aromatic cyclic group, e.g., in certainembodiments, R^(B3) is optionally substituted aryl or optionallysubstituted heteroaryl.

In certain embodiments, R^(B3) is optionally substituted carbocyclyl,e.g., optionally substituted C₃₋₆carbocyclyl. In certain embodimentsR^(B3) is optionally substituted C₃ carbocyclyl, optionally substitutedC₄ carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl. In certain embodiments, R^(B3) is optionallysubstituted cyclopenyl (C₅) or optionally substituted cyclohexyl (C₆).

In certain embodiments, R^(B3) is optionally substituted carbocyclylfused to an optionally substituted aryl or optionally substitutedheteroaryl ring, wherein the point of attachment is on the carbocyclylring. In certain embodiments, R^(B3) is optionally substitutedcarbocyclyl, e.g., optionally substituted C₃₋₆carbocyclyl, fused to anoptionally substituted aryl or optionally substituted heteroaryl ring.In certain embodiments R^(B3) is optionally substituted C₃ carbocyclyl,optionally substituted C₄ carbocyclyl, optionally substituted (C₅)carbocyclyl, or optionally substituted C₆ carbocyclyl, fused to anoptionally substituted aryl or optionally substituted heteroaryl ring.In certain embodiments, R^(B3) is optionally substituted cyclopenyl (C₅)or optionally substituted cyclohexyl (C₆) fused to an optionallysubstituted aryl or optionally substituted heteroaryl ring. In certainembodiments, the fused aryl ring is an optionally substituted phenylring. In certain embodiments, the fused heteroaryl ring is an optionallysubstituted 6-membered heteroaryl ring, e.g., an optionally substitutedpyridinyl ring.

In certain embodiments, R^(B3) is optionally substituted heterocyclyl,e.g., optionally substituted 3-6 membered heterocyclyl. In certainembodiments R^(B3) is optionally substituted 3-membered heterocyclyl,optionally substituted 4-membered heterocyclyl, optionally substituted5-membered heterocyclyl, or optionally substituted 6-memberedheterocyclyl, e.g., comprising 1 or 2 heteroatoms selected from oxygen,sulfur, or nitrogen.

In certain embodiments, R^(B3) is optionally substituted heterocyclylfused to an optionally substituted aryl or optionally substitutedheteroaryl ring, wherein the point of attachment is on the heterocyclylring. In certain embodiments, R^(B3) is optionally substitutedheterocyclyl, e.g., optionally substituted 3-6 membered heterocyclyl,fused to an optionally substituted aryl or optionally substitutedheteroaryl ring. In certain embodiments R^(B3) is optionally substituted3-membered heterocyclyl, optionally substituted 4-membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl, e.g., comprising 1 or 2 heteroatomsselected from oxygen, sulfur, or nitrogen, fused to an optionallysubstituted aryl or optionally substituted heteroaryl ring. In certainembodiments, the fused aryl ring is a fused optionally substitutedphenyl ring. In certain embodiments, the fused heteroaryl ring is a6-membered heteroaryl ring, e.g., an optionally substituted pyridinylring. In certain embodiments, the point of attachment of R^(B3) is via anitrogen atom. In certain embodiments, R^(B3) is an optionallysubstituted 1,2,3,4-tetrahydro-2,7-naphthyridinyl ring, a3,4-dihydropyrido[4,3-d]pyrimidinyl ring, a3,4-dihydropyrido[4,3-d]pyrimidin-2-one ring, or a3,4-dihydro-2H-pyrido[3,4-e][1,3]oxazin-2-one ring, wherein the point ofattachment is on the non-aromatic heterocyclyl ring.

In certain embodiments, R^(B3) is optionally substituted aryl, e.g.,optionally substituted C₆₋₁₄aryl. In certain embodiments, R^(B3) isoptionally substituted phenyl. In certain embodiments, R^(B3) isoptionally substituted naphthyl. In certain embodiments, R^(B3) isoptionally substituted phenyl fused to an optionally substitutedheterocyclyl ring; such as an optionally substituted phenyltetrahydroisoquinolinyl. It is understood in reference to optionallysubstituted aryl ring systems comprising a fused heterocyclyl ring thatthe point of attachment to the parent molecule is on the aryl (e.g.,phenyl) ring.

In certain embodiments, R^(B3) is optionally substituted heteroaryl,e.g., optionally substituted 5-14 membered heteroaryl. In certainembodiments, R^(B3) is an optionally substituted 5-membered heteroarylor an optionally substituted 6-membered heteroaryl. In certainembodiments, R^(B3) is an optionally substituted bicyclic heteroaryl,e.g., an optionally substituted 5,6-bicyclic heteroaryl, or optionallysubstituted 6,6-bicyclic heteroaryl. In certain embodiments, R^(B3) isan optionally substituted 5,6-bicyclic heteroaryl or optionallysubstituted 6,6-bicyclic heteroaryl ring system selected from the groupconsisting of optionally substituted naphthyridinyl, optionallysubstituted pteridinyl, optionally substituted quinolinyl, optionallysubstituted isoquinolinyl, optionally substituted cinnolinyl, optionallysubstituted quinoxalinyl, optionally substituted phthalazinyl, andoptionally substituted quinazolinyl. In certain embodiments, the pointof attachment of R^(B3) is via a nitrogen atom.

In certain embodiments, wherein R^(B3) is an optionally substitutedheterocyclyl, -L₁-R^(B3) is selected from the group consisting of:

wherein:

each instance of R^(6A) is independently halogen, —NO₂, —CN, —OR^(6C),—SR^(6C), —N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C), —C(═O)N(R^(6C))₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl;

each instance of R^(6B) is independently hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group when attached tonitrogen;

wherein each instance of R^(6C) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached tooxygen, a sulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen, optionally when attached toN the two R^(6C) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring; and

m is 0 or an integer between 1 and 4, inclusive,

provided L¹ is not —N(R^(L))—(C(R^(LL))₂)_(p)— wherein p is 0.

In certain embodiments, wherein R^(B3) is an optionally substituted arylor optionally substituted heteroaryl, -L₁-R^(B3) is selected from thegroup consisting of:

wherein:

each instance of R^(6A) is independently halogen, —NO₂, —CN, —OR^(6C),—SR^(6C), —N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C), —C(═O)N(R^(6C))₂,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl;

each instance of R^(6B) is independently hydrogen, optionallysubstituted alkyl, or a nitrogen protecting group when attached tonitrogen;

wherein each instance of R^(6C) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached tooxygen, a sulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen, optionally when attached toN the two R^(6C) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring; and

m is 0 or an integer between 1 and 4, inclusive.

In certain embodiments, m is 0. In certain embodiments, m is 1, 2, 3, or4. In certain embodiments, wherein m is 1, 2, 3, or 4, at least oneR^(6A) is halogen (e.g., fluoro), —OR^(6C), —SR^(6C), or —N(R^(6C))₂.

In certain embodiments, L₁ is a bond or —C(═O)N(R^(L))—, wherein R^(L)is hydrogen or an optionally substituted alkyl (e.g., methyl), andR^(B3) is optionally substituted aryl or optionally substitutedheteroaryl, as described herein.

For example, in certain embodiments of Formula (A), (B), (C), (D), and(E), wherein the group -L₁-R^(B3) is a group of formula:

wherein L₁ is a bond, provided are compounds of Formula (A-h), (A-1-h),(A-2-h), (B-h), or (C-h), (D-h), (D-1-h), (D-2-h), (E-h), (E-1-h),(E-2-h):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(6A), and m are as defined herein.

For example, in certain embodiments of Formula (A), (B), and (C) whereinthe group -L₁-R^(B3) is a group of formula:

wherein L₁ is a bond, provided are compounds of Formula (A-i), (A-1-i),(A-2-i), (B-i), or (C-i):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(6A), and m are as defined herein.

In certain embodiments of Formula (A), (B), and (C), wherein the group-L₁-R^(B3) is a group of formula:

wherein L₁ is a bond, provided are compounds of Formula (A-p), (A-1-p),(A-2-p), (B-p), or (C-p):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(6A), and m are as defined herein.

In certain embodiments of Formula (A), (B), and (C), wherein the group-L₁-R^(B3) is a group of formula:

wherein L₁ is a bond, provided are compounds of Formula (A-q), (A-1-q),(A-2-q), (B-q), or (C-q):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, wherein

, R¹, R², R³, R⁴, R⁵, R^(6A), and m are as defined herein.

In certain embodiments of Formula (A), (B), and (C), wherein the group-L₁-R^(B3) is a group of formula:

wherein L₁ is a bond, provided are compounds of Formula (A-r), (A-1-r),(A-2-r), (B-r), or (C-r):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(6A), and m are as defined herein.

Compounds of Formula (A-i), (A-1-i), (A-2-i), (B-i), (C-i), (A-p),(A-1-p), (A-2-p), (B-p), (C-p), (A-q), (A-1-q), (A-2-q), (B-q), (C-q),(A-r), (A-1-r), (A-2-r), (B-r), and (C-r) are designed to mimic theisoquinoline of cortistatin A and are envisioned to have improvedmetabolic stability.

In other embodiments of Formula (A), (B), and (C), wherein R^(B1) is-L₁-R^(B3), and L₁ is —C(═O)N(R^(L))—, provided are compounds of Formula(A-j), (A-1-j), (A-2-j), (B-j), or (C-j):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(L), and R^(B3) are as defined herein.

In other embodiments of Formula (A), (B), and (C), wherein R^(B1) is-L₁-R^(B3), and L₁ is —N(R^(L))—(C(R^(LL))₂)_(p)—, wherein p is 1,provided are compounds of Formula (A-n), (A-1-n), (A-2-n), (B-n), or(C-n):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, R^(L), and R^(B3) are as defined herein.

Compounds of Formula (A-j), (A-1-j), (A-2-j), (B-j), (C-j), (A-n),(A-1-n), (A-2-n), (B-n), and (C-n) are designed to mimic theisoquinoline of cortistatin A and are envisoned to have improvedpharmacokinetic properties. In particular, compounds wherein -L₁-R^(B3)is a group of formula:

wherein L₁ is —C(O)N(R^(L))— or —N(R^(L))—((CR^(LL))₂)_(p)—, wherein pis 1, are contemplated herein.

Alternatively, in certain embodiments, at least one instance of R^(B1)and R^(B2) is —X^(A)R^(A). In this instance, in certain embodiments, theother of R^(B1) and R^(B2) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)).For example, in certain embodiments, when

designated as (c) represents a single bond, then R^(B1) is —X^(A)R^(A)(e.g., —OR^(A)) and R^(B2) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)).In other embodiments, when

designated as (c) represents a single bond, then R^(B2) is —X^(A)R^(A)(e.g., —OR^(A)) and R^(B1) is hydrogen or —X^(A)R^(A) (e.g., —OR^(A)).Alternatively, in certain embodiments, when

designated as (c) represents a double bond, then R^(B1) is —X^(A)R^(A)(e.g., —OR^(A) wherein R^(A) is not hydrogen) and R^(B2) is absent.

In certain embodiments, both instances of R^(B1) and R^(B2) is—X^(A)R^(A). In this embodiments, in certain instances, the two R^(A)groups may be joined to form an optionally substituted heterocyclylring, e.g., an optionally substituted 5-6 membered heterocyclyl ring.For example, in certain embodiments of Formula (A), (B), and (C),wherein both instances of R^(B)1 and R^(B2) is —X^(A)R^(A), provided arecompounds of Formula (A-k), (A-1-k), (A-2-k), and (B-k), and (C-k):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, R⁵, X^(A) and R^(A), are as defined herein.

In certain embodiments, R^(B1) and R^(B2) are joined to form an oxogroup (═O). For example, in certain embodiments of Formula (A), (B), and(C), R^(B1) and R^(B2) are joined to form an oxo group (═O), providedare compounds of Formula (A-m), (A-1-m), (A-2-m), (B-m), or (C-m):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R³, R⁴, and R⁵, are as defined herein.

Exemplary Compounds

Various combinations of certain embodiments are further contemplatedherein.

For example, in certain embodiments of Formula (A-1-h), (A-1-i),(A-1-p), (A-1-q), (A-1-r), (A-1-j), (A-1-n), (A-2-h), (D-1-h), (D-2-h),(E-1-h), (E-2-h), and wherein the bond

designated as (c) is a single bond, and R^(B2) is hydrogen, provided isa compound of Formulae:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein

, R¹, R², R^(O), R³, R⁴, R⁵, R^(6A), R^(6B), R^(L), R^(LL), and m are asdefined herein. In certain embodiments, R³ is methyl. In certainembodiments, R⁴ is hydrogen. In certain embodiments, R⁵ is hydrogen. Incertain embodiments the bonds

designated as (a) and (b) are double bonds. In certain embodiments, eachinstance of R¹ and R² is optionally substituted alkyl, e.g., —CH₃. Incertain embodiments, at least one of R¹ and R² is a group of formula

wherein p and Z are as defined herein. In certain embodiments, at leastone of R¹ and R² is —S(═O)₂R^(A). In certain embodiments, R¹ and R² arejoined to form an optionally substituted heterocyclyl. In certainembodiments, R¹ and R² are joined to form a ring of formula:

wherein R⁷, n and G are as defined herein. In certain embodiments, thecompound is an N-oxide, e.g., the group

is of formula

In certain embodiments, R^(O) is an optionally substituted alkyl, e.g.,—CH₃ or a group of formula

wherein Z and p are as defined herein. In certain embodiments, R^(O) is—C(═O)R^(A), —C(═O)OR^(A), or —C(═O)N(R^(A))₂, e.g., —C(═O)CH₃,—C(═O)OCH₃, —C(═O)N(CH₃)₂, or —C(═O)NHCH₃.

In certain embodiments of Formula (A-1-h-i), (A-1-i-i), (A-1-p-i),(A-1-q-i), (A-1-r-i), (A-1-j-i), (A-1-n-i), (A-2-h-i), (D-1-h-i),(D-2-h-i), (E-1-h-i), and (E-2-h-i), wherein R³ is methyl, R⁴ and R⁵ arehydrogen, and the bonds designated as (a) and (b) are double bonds,provided is a compound of Formulae:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R¹, R², R^(O), R^(6A), R^(6A), R^(6B), R^(L), R^(LL)and m are as defined herein. In certain embodiments, each instance of R¹and R² is optionally substituted alkyl, e.g., —CH₃. In certainembodiments, one of R¹ and R² is hydrogen, and one of R¹ and R² isoptionally substituted alkyl, e.g., —CH₃. In certain embodiments, atleast one of R¹ and R² is a group of formula

wherein p and Z are as defined herein. In certain embodiments, at leastone of R¹ and R² is —S(═O)₂R^(A). In certain embodiments, R¹ and R² arejoined to form an optionally substituted heterocyclyl. In certainembodiments, R¹ and R² are joined to form a ring of formula:

wherein R⁷, n and G are as defined herein. In certain embodiments, thecompound is an N-oxide, e.g., the group

is of formula

In certain embodiments, R^(O) is an optionally substituted alkyl, e.g.,—CH₃ or a group of formula

wherein Z and p are as defined herein. In certain embodiments, R^(O) is—C(═O)R^(A), —C(═O)OR^(A), or —C(═O)N(R^(A))₂, e.g., —C(═O)CH₃,—C(═O)OCH₃, —C(═O)N(CH₃)₂, or —C(═O)NHCH₃.

In certain embodiments of Formula (A-1-h-ii) and (A-2-h-ii), wherein R¹and R² are joined to form an optionally substituted heterocyclyl,provided is a compound of Formula:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R⁷, R^(6A), n and m are as defined herein. In certainembodiments, G is O. In certain embodiments, G is N—CH₃. In certainembodiments, m is 0. In certain embodiments, m is 1. In certainembodiments, n is 0. In certain embodiments, n is 1.

In certain embodiments of Formula (A-1-h-ii) or (A-2-h-ii), wherein R¹and R² are joined to form an optionally substituted heterocyclyl,provided is a compound of Formula:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R⁷, R^(6A), n and m are as defined herein. In certainembodiments, G is —CH₂—. In certain embodiments, m is 0. In certainembodiments, m is 1. In certain embodiments, n is 0. In certainembodiments, n is 1.

In certain embodiments of Formula (A-1-h-ii), wherein each of R¹ and R²are —CH₃, provided is a compound of Formula:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R^(6A), and m are as defined herein. In certainembodiments, m is 0. In certain embodiments, m is 1.

In certain embodiments of Formula (A-2-h-ii), wherein one of R¹ and R²is hydrogen, and the other of R¹ and R² is —CH₃, provided is a compoundof Formula:

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; wherein R^(6A), and m are as defined herein. In certainembodiments, m is 0. In certain embodiments, m is 1.

Exemplary compounds of Formula (A) include, but are not limited to:

and pharmaceutically acceptable salts, quaternary amine salts thereof,and N-oxides thereof, e.g., N-oxides of formula:

However, in certain embodiments, the compound of Formula (A) is not:

or a pharmaceutically acceptable salt or quaternary amine salt thereof,or an N-oxide thereof.

Exemplary compounds of Formula (B) include, but are not limited to:

and pharmaceutically acceptable salts, quaternary amine salts, orN-oxides thereof.

Exemplary compounds of Formula (C) include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of Formula (D) include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of Formula (E) include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising a compound of Formula (A), (B), (C), (D), or (E)or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof, and a pharmaceutically acceptable excipient. In certainembodiments, the compound is present in an effective amount, e.g., atherapeutically effective amount or a prophylactically effective amount.

Pharmaceutically acceptable excipients include any and all solvents,diluents or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. General considerations in theformulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21^(st) Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing the compound of Formula (A), (B),(C), (D), or (E) or a pharmaceutically acceptable salt, quaternary aminesalt, or N-oxide thereof (the “active ingredient”) into association withthe excipient and/or one or more other accessory ingredients, and then,if necessary and/or desirable, shaping and/or packaging the product intoa desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, etc., and combinationsthereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g. acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g.bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]),long chain amino acid derivatives, high molecular weight alcohols (e.g.stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate,ethylene glycol distearate, glyceryl monostearate, and propylene glycolmonostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene,polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer),carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium,powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acidesters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20],polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate[Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate[Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitanmonooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylenemonostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,polyethoxylated castor oil, polyoxymethylene stearate, and Solutol),sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.,Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether[Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate,oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68,Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride,benzalkonium chloride, docusate sodium, etc. and/or combinationsthereof.

Exemplary binding agents include starch (e.g. cornstarch and starchpaste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghattigum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum),and larch arabogalactan), alginates, polyethylene oxide, polyethyleneglycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes,water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, etc., and combinations thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, andcombinations thereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the activeingredient(s), the liquid dosage forms may comprise inert diluentscommonly used in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, andperfuming agents. In certain embodiments for parenteral administration,the conjugates of the invention are mixed with solubilizing agents suchas Cremophor, alcohols, oils, modified oils, glycols, polysorbates,cyclodextrins, polymers, polymer conjugates (e.g., IT-101/CLRX101), andcombinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of the active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the active ingredientthen depends upon its rate of dissolution which, in turn, may dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered form is accomplished bydissolving or suspending the active ingredient in an oil vehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active ingredient(s) can be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compoundof this invention may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, theactive ingredient is admixed under sterile conditions with apharmaceutically acceptable carrier and/or any needed preservativesand/or buffers as can be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of an active ingredient tothe body. Such dosage forms can be prepared, for example, by dissolvingand/or dispensing the active ingredient in the proper medium.Alternatively or additionally, the rate can be controlled by eitherproviding a rate controlling membrane and/or by dispersing the activeingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionscan be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition can be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 toabout 7 nanometers or from about 1 to about 6 nanometers. Suchcompositions are conveniently in the form of dry powders foradministration using a device comprising a dry powder reservoir to whicha stream of propellant can be directed to disperse the powder and/orusing a self propelling solvent/powder dispensing container such as adevice comprising the active ingredient dissolved and/or suspended in alow-boiling propellant in a sealed container. Such powders compriseparticles wherein at least 98% of the particles by weight have adiameter greater than 0.5 nanometers and at least 95% of the particlesby number have a diameter less than 7 nanometers. Alternatively, atleast 95% of the particles by weight have a diameter greater than 1nanometer and at least 90% of the particles by number have a diameterless than 6 nanometers. Dry powder compositions may include a solid finepowder diluent such as sugar and are conveniently provided in a unitdose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations can be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 to 500 micrometers. Such aformulation is administered by rapid inhalation through the nasalpassage from a container of the powder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) and as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A pharmaceutical composition of the invention can beprepared, packaged, and/or sold in a formulation for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and maycontain, for example, 0.1 to 20% (w/w) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations for buccal administration may comprise apowder and/or an aerosolized and/or atomized solution and/or suspensioncomprising the active ingredient. Such powdered, aerosolized, and/oratomized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 to about 200 nanometers,and may further comprise one or more of the additional ingredientsdescribed herein.

A pharmaceutical composition can be prepared, packaged, and/or sold in aformulation for ophthalmic administration. Such formulations may, forexample, be in the form of eye drops including, for example, a 0.1/1.0%(w/w) solution and/or suspension of the active ingredient in an aqueousor oily liquid carrier. Such drops may further comprise bufferingagents, salts, and/or one or more other of the additional ingredientsdescribed herein. Other opthalmically administrable formulations whichare useful include those which comprise the active ingredient inmicrocrystalline form and/or in a liposomal preparation. Ear dropsand/or eye drops are contemplated as being within the scope of thisinvention.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.General considerations in the formulation and/or manufacture ofpharmaceutical compositions can be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Still further encompassed by the invention are pharmaceutical packsand/or kits. Pharmaceutical packs and/or kits provided may comprise aprovided composition and a container (e.g., a vial, ampoule, bottle,syringe, and/or dispenser package, or other suitable container). In someembodiments, provided kits may optionally further include a secondcontainer comprising a suitable aqueous carrier for dilution orsuspension of the provided composition for preparation of administrationto a subject. In some embodiments, contents of provided formulationcontainer and solvent container combine to form at least one unit dosageform.

Optionally, a single container may comprise one or more compartments forcontaining a provided composition, and/or appropriate aqueous carrierfor suspension or dilution. In some embodiments, a single container canbe appropriate for modification such that the container may receive aphysical modification so as to allow combination of compartments and/orcomponents of individual compartments. For example, a foil or plasticbag may comprise two or more compartments separated by a perforated sealwhich can be broken so as to allow combination of contents of twoindividual compartments once the signal to break the seal is generated.A pharmaceutical pack or kit may thus comprise such multi-compartmentcontainers including a provided composition and appropriate solventand/or appropriate aqueous carrier for suspension.

Optionally, instructions for use are additionally provided in such kitsof the invention. Such instructions may provide, generally, for example,instructions for dosage and administration. In other embodiments,instructions may further provide additional detail relating tospecialized instructions for particular containers and/or systems foradministration. Still further, instructions may provide specializedinstructions for use in conjunction and/or in combination withadditional therapy.

Methods of Treatment

CDK8 and CDK19, referred to as “Mediator kinases”, assemble inmulti-protein complexes that reversibly bind the Mediator complex. TheMediator complex links enhancer-bound transcription factors topromoter-bound RNA pol II holoenzyme and it influences chromatinarchitecture to regulate transcription and gene expression through stillpoorly understood mechanisms. Recent comprehensive genome-widesequencing of samples from 200 AML patients revealed that, remarkably,nearly all mutations in presumably cancer-driving proteins areassociated with regulating gene expression. See, e.g., Aerts, et al.,Nature (2013) 499:35-36; The Cancer Genome Atlas Research Network, 2013.Genomic and Epigenomic Landscapes of Adult De Novo Acute MyeloidLeukemia. N. Engl. J. Med. 368, 2059-2074. Therefore, specificinhibition of Mediator kinases might be a new means to disrupt theability of some AML mutations to deregulate gene expression programsthat drive AML cell growth. Specific small molecule inhibition ofCDK8/CDK19 may also prove beneficial for treating other cancers thatrely on deregulated gene expression. CDK8/cyclin C was further observedto be more highly expressed in neurons and astrocytes of Alzheimer'sdisease (AD) patients, and thus specific small molecule inhibition ofCDK8 may also prove beneficial for treating degenerative disorders, suchas AD. See, e.g., Hessel et al., Neurobiology of Aging (2003)24:427-435, wherein. Cortistatin A has been reported to bind to CDK8 andCDK19. See, e.g., Cee et al., Angew Chem Int Ed (2009) 48:8952 and US20120071477. Furthermore, as FIGS. 7, 10A-10C, and 16A-16D demonstrate,cortistatin A inhibits CDK8 kinase activity, in part due to thisbinding. CDK8 and CDK19 have very similar sequences and catalyticdomains suggesting that inhibiting CDK8 will likely also inhibit CDK19.See, e.g., Ries et al., Semin. Cell Dev. Biol. (2011) 22:735-740. Blastalignment of CDK8 vs. CDK19 also indicate that the amino acids are 70%identical and 82% similar.

Thus, in one aspect, provided is a method of inhibiting CDK8 and/orCDK19 kinase activity in a cell comprising contacting a compound ofFormula (A), (B), (C), (D), or (E) or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof, with the cell. In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method. In another aspect, provided is a methodof treating a condition associated with CDK8 and/or CDK19 kinaseactivity, comprising administering to a subject in need thereof acompound Formula (A), (B), (C), (D), or (E) or a pharmaceuticallyacceptable salt, quaternary amine salt, or N-oxides thereof.

In certain embodiments, the condition associated with CDK8 and/or CDK19kinase activity is a proliferative disorder, e.g., cancer. CDK8 kinaseactivity has been linked to colon cancer. See, e.g., Firestein, et al.,Nature (2008) 455:547-551.

In certain embodiments, the condition associated with CDK8 and/or CDK19kinase activity is a diabetic condition, e.g., diabetes.

In certain embodiments, the condition associated with CDK8 and/or CDK19kinase activity is a degenerative disorder, e.g., Alzheimer's disease(AD).

CDK8 has been linked to regulation of a number of signaling pathways andtranscriptional programs that have been implicated in maintaining anddriving diseases such as cancer. These pathways and programs includeWnt/beta-catenin pathway, Notch pathway, TGF-beta/BMP signaling,JAK-STAT pathway, p53 pathway, and hypoxia response. AberrantWnt/beta-catenin signaling is associated with leukemias and many othercancers. For instance, the most common mutations in colon cancer areones that lead to activation of Wnt/beta-catenin signaling, expressionof Wnt-target genes, and tumorigenesis. Given its central role intumorigenesis, there is much interest in identifying safe, effectiveinhibitors of Wnt/beta-catenin signaling. See, e.g., Wang, et al.,Science (2010) 327:1650-1653. Polakis, EMBO J. (2012) 31: 2737-2746.

Thus, in another aspect, provided is a method of treating a β-cateninpathway-associated condition comprising administering to a subject inneed thereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof.In another aspect, provided is a method of modulating the β-cateninpathway (e.g., by inhibiting the expression of beta-catenin targetgenes) in a cell comprising contacting a compound of Formula (A), (B),(C), (D), or (E) or a pharmaceutically acceptable salt, quaternaryamine, or N-oxide thereof, with the cell. In certain embodiments, themethod is an in vitro method. In certain embodiments, the method is anin vivo method.

As shown in FIG. 15, cortistatin A inhibits beta-catenin activatedtranscription in a reporter gene assay and expression of putativeWnt/beta-catenin target genes in AML cells. Numerous studies have linkedbeta-catenin pathway activation to tumor initiation, maintenance, andgrowth. Wnt/beta-catenin pathway alterations have been observed inbreast cancer, colorectal cancer, hepatocellular carcinoma,medulloblastoma, pancreatic cancer, lymphoma/leukemia, lung cancer,kidney cancer, and Wilms' tumor. See, e.g., Saito-Diaz, et al., GrowthFactors (2013) 31:1-31. In addition to cancer, other diseases withoveractivation of the Wnt/beta-catenin pathway include high bone massdiseases and hypertrophic obesity. Furthermore, variants of the Wnt-betacatenin pathway transcription factor TCF7L2 have been associated withdiabetes. See, e.g., MacDonald et al., Developmental Cell (2009) 17,9-26.

In another aspect, provided is a method of treating a JAK-STATpathway-associated condition comprising administering to a subject inneed thereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof.In another aspect, provided is a method of modulating the STAT1 activityin a cell (e.g., by inhibiting phosphorylation of STAT1 S727 in theJAK-STAT pathway, leading to up- or down-regluation of specificSTAT1-associated genes) comprising contacting a compound of Formula (A),(B), (C), (D), or (E) or a pharmaceutically acceptable salt, quaternaryamine, or N-oxide thereof, with the cell. In certain embodiments, themethod is an in vitro method. In certain embodiments, the method is anin vivo method.

As shown in FIG. 7, cortistatin A inhibits interferon-gamma-stimulatedSTAT1 phosphorylation. Inhibition of STAT1 phosphorylation may be atherapeutic strategy to treat aberrant inflammation, including inatherosclerosis, to treat cancers, including MPNs and leukemias, and totreat diabetes, through prevention of STAT1-mediated beta-cellapoptosis. IFN-gamma is expressed at high levels in atheroscleroticlesions leading to increased inflammation through STAT1 activation andIFN-gamma activates STAT1 to induce beta-cell apoptosis. See, e.g.,Gysemans et al., Biochem. Soc. Trans (2008) 36:328. Phosphorylation ofSTAT1 by CDK8 has also been shown to restrain NK activation and tumorsurvellience. Therefore, inhibition of CDK8 kinase activity maybeneficially enable an NK-mediated tumor cell killing in addition todirectly inhibiting the proliferation of tumor cells. See, e.g., Putz etal., Cell Reports (2013) 4:437-444.

It has been reported that nuclear CDKs, such as CDK8, drive SMADtranscriptional activation and turnover in BMP and TGF-beta. See, e.g.,Alarcon et al., Cell (2009) 139:757-769. Thus, in yet another aspect,provided is a method of treating a TGF-beta/BMP pathway-associatedcondition comprising administering to a subject in need thereof acompound of Formula (A), (B), (C), (D), or (E) or a pharmaceuticallyacceptable salt, quaternary amine, or N-oxide thereof. In anotheraspect, provided is a method of modulating the TGF-beta/BMP pathway(e.g., by inhibiting CDK8/CDK19 phosphorylation SMAD proteins in theTGF-beta/BMP pathway leading to up- or down-regulation of specific SMADprotein-associated genes) in a cell comprising contacting a compound ofFormula (A), (B), (C), (D), or (E) or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof, with the cell. In certainembodiments, the method is an in vitro method. In certain embodiments,the method is an in vivo method.

TGF-beta and BMP pathways are critical for tissue homeostasis,modulation of TGF-beta and BMP pathway activity may be a treatmentstrategy for conditions including but not limited to muscle dystrophy(see, e.g., Ceco, FEBS J. (2013) 280:4198-4209), immune response totransplants, cancer, fibrosis, and Marfan syndrome (see, e.g., Akhurstand Hata, Nat Rev Drug Discov (2012) 11:790-811).

Hypoxia is a condition in which the body or region of the body isdeprived of adequate oxygen supply, and may result from altitudesickness, ischaemia, stroke, heart attack, anemia, cancer, and carbonmonoxide poisoning. CDK8 has been linked to regulation of hypoxicresponse, playing a role in induction of HIF-1-A (HIF-1-alpha) targetgenes. These genes are involved in angiogenesis, glycolysis, metabolicadaption, and cell survival, processes critical to tumor maintenance andgrowth. See, e.g., Galbraith, et al., Cell 153:1327-1339.

Thus, in one aspect, provided is a method of treating a conditionassociated with hypoxia comprising administering to a subject in needthereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof.In another aspect, provided is a method of reducing hypoxia injurycomprising administering to a subject in need thereof a compound ofFormula (A), (B), (C), (D), or (E) or a pharmaceutically acceptablesalt, quaternary amine, or N-oxide thereof. In yet another aspect,provided is a method of modulating HIF-1-A (HIF-1-alpha) activity (e.g.,by inhibiting the expression HIF-1-alpha associated genes) in a cellcomprising contacting a compound of Formula (A), (B), (C), (D), or (E)or a pharmaceutically acceptable salt, quaternary amine, or N-oxidethereof, with the cell. In certain embodiments, the method is an invitro method. In certain embodiments, the method is an in vivo method.

In another aspect, provided is a method of increasing BIM expression(e.g., BCLC2L11 expression) to induce apoptosis in a cell comprisingcontacting a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof,with the cell. In certain embodiments, the method is an in vitro method.In certain embodiments, the method is an in vivo method. BCL2L11expression is tightly regulated in a cell. BCL2L11 encodes for BIM, aproapoptotic protein. BCL2L11 is downregulated in many cancers and BIMis inhibited in many cancers, including chronic myelocytic leukemia(CML) and non-small cell lung cancer (NSCLC) and that suppression ofBCL2L11 expression can confer resistance to tyrosine kinase inhibitors.See, e.g., Ng et al., Nat. Med. (2012) 18:521-528.

Furthermore, the cortistatins as a class of compounds have been found tohave anti-antiogenic activity. See, e.g., Aoki, et al., JACS (2006) 128:3148-9. Angiogenesis is the process of generating new capillary bloodvessels from the pre-existing vasculature. After birth, angiogenesiscontributes to organ growth, but in adulthood it is strictly regulatedand occurs only during wound healing and in the female reproductivecycle. See, e.g., Klagsbrun et al., Molecular angiogenesis. Chemistry &Biology 1999, 6 (8), R217-R224. Under normal physiological conditions,angiogenesis is tightly controlled by a series of pro-angiogenic andanti-angiogenic factors, which allow vascular growth for controlledperiods of time. See, e.g., Ferrara, Vascular Endothelial Growth Factoras a Target for Anticancer Therapy. The Oncologist 2004, 9:2-10.Persistent, unregulated angiogenesis has been implicated in a wide rangeof diseases, including rheumatoid arthritis, macular degeneration,atherosclerosis, obesity, benign neoplasms, and cancers. See, e.g.,Moulton et al., Angiogenesis inhibitors endostatin or TNP-470 reduceintimal neovascularization and plaque growth in apolipoproteinE-deficient mice. Circulation 1999, 99, (13), 1726-1732; and Hanahan etal., The hallmarks of cancer. Cell 2000, 100, (1), 57-70. That thesepathological states are unified by their status as“angiogenesis-dependent diseases” but are otherwise unrelated has ledFolkman to propose the concept of angiogenesis as an “organizingprinciple” in biology, by which many types of seemingly dissimilarphenomena may be connected. See Folkman, Opinion-Angiogenesis: anorganizing principle for drug discovery? Nature Reviews Drug Discovery2007, 6(4):273-286.

Thus, in yet another aspect, provided is a method of treating acondition associated with angiogenesis, such as, for example, a diabeticcondition (e.g., diabetic retinopathy), an inflammatory condition (e.g.,rheumatoid arthritis), macular degeneration, obesity, atherosclerosis,or a proliferative disorder, comprising administering to a subject inneed thereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof.

In certain embodiments, the condition associated with angiogenesis is adiabetic condition or associated complication. In certain embodiments,provided is a method of treating a diabetic condition or associatedcomplication comprising administering to a subject in need thereof acompound of Formula (A), (B), (C), (D), or (E) or a pharmaceuticallyacceptable salt, quaternary amine salt, or N-oxide thereof.

As used herein, a “diabetic condition” refers to diabetes andpre-diabetes. Diabetes refers to a group of metabolic diseases in whicha person has high blood sugar, either because the body does not produceenough insulin, or because cells do not respond to the insulin that isproduced. This high blood sugar produces the classical symptoms ofpolyuria (frequent urination), polydipsia (increased thirst) andpolyphagia (increased hunger). There are several types of diabetes. TypeI diabetes results from the body's failure to produce insulin, andpresently requires the person to inject insulin or wear an insulin pump.Type 2 diabetes results from insulin resistance a condition in whichcells fail to use insulin properly, sometimes combined with an absoluteinsulin deficiency. Gestational diabetes occurs when pregnant womenwithout a previous diagnosis of diabetes develop a high blood glucoselevel. Other forms of diabetes include congenital diabetes, which is dueto genetic defects of insulin secretion, cystic fibrosis-relateddiabetes, steroid diabetes induced by high doses of glucocorticoids, andseveral forms of monogenic diabetes, e.g., mature onset diabetes of theyoung (e.g., MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Pre-diabetesindicates a condition that occurs when a person's blood glucose levelsare higher than normal but not high enough for a diagnosis of diabetes.

All forms of diabetes increase the risk of long-term complications(referred to herein as the “associated complication” of the diabeticcondition). These typically develop after many years, but may be thefirst symptom in those who have otherwise not received a diagnosisbefore that time. The major long-term complications relate to damage toblood vessels. Diabetes doubles the risk of cardiovascular disease andmacrovascular diseases such as ischemic heart disease (angina,myocardial infarction), stroke, and peripheral vascular disease.Diabetes also causes microvascular complications, e.g., damage to thesmall blood vessels. Diabetic retinopathy, which affects blood vesselformation in the retina of the eye, can lead to visual symptoms, reducedvision, and potentially blindness. Diabetic nephropathy, the impact ofdiabetes on the kidneys, can lead to scarring changes in the kidneytissue, loss of small or progressively larger amounts of protein in theurine, and eventually chronic kidney disease requiring dialysis.Diabetic neuropathy is the impact of diabetes on the nervous system,most commonly causing numbness, tingling and pain in the feet and alsoincreasing the risk of skin damage due to altered sensation. Togetherwith vascular disease in the legs, neuropathy contributes to the risk ofdiabetes-related foot problems, e.g., diabetic foot ulcers, that can bedifficult to treat and occasionally require amputation.

As will be appreciated by those of ordinary skill in this art, intreating a diabetic condition or complication, an effective amount of acompound administered may, for example, reduce, prevent, or delay theonset, of any one of the following symptoms: reduce fasting plasmaglucose level [typical diabetic level is ≧7.0 mmol/l (126 mg/dl);typical prediabetic range is 6.1 to 6.9 mmol/1]; reduce plasma glucose[typical diabetic level is ≧11.1 mmol/l (200 mg/dL) two hours after a 75g oral glucose load as in a glucose tolerance test]; reduce symptoms ofhyperglycemia and casual plasma glucose [typical diabetic level is ≧11.1mmol/l (200 mg/dl)]; reduce levels of glycated hemoglobin (Hb A1C)[typical diabetic level is ≧6.5%]. Subjects with fasting glucose levelsfrom 110 to 125 mg/dl (6.1 to 6.9 mmol/1) are considered to haveimpaired fasting glucose. Subjects with plasma glucose at or above 140mg/dL (7.8 mmol/L), but not over 200 mg/dL (11.1 mmol/L), two hoursafter a 75 g oral glucose load are considered to have impaired glucosetolerance.

In certain embodiments, the associated complication is diabeticretinopathy. For example, in certain embodiments, provided is a methodof treating diabetic retinopathy comprising administering to a subjectin need thereof a compound of Formula (A), (B), (C), (D), or (E), or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof.

In certain embodiments, the condition associated with angiogenesis ismacular degeneration. In certain embodiments, provided is a method oftreating macular degeneration comprising administering to a subject inneed thereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof.

In certain embodiments, the condition associated with angiogenesis isobesity. As used herein, “obesity” and “obese” as used herein, refers toclass I obesity, class II obesity, class III obesity and pre-obesity(e.g., being “over-weight”) as defined by the World Health Organization.In certain embodiments, provided is a method of treating obesitycomprising administering to a subject in need thereof a compound ofFormula (A), (B), (C), (D), or (E) or a pharmaceutically acceptablesalt, quaternary amine salt, or N-oxide thereof. Evidence suggests thatadipose tissue expansion is dependent on vasculature development.Therefore, inhibition of angiogenesis may be therapeutic strategy forrestricting the expansion of adipose tissue to prevent and treatobesity. See, e.g., Christiaens and Lijnen, Molecular and CellularEndocrinology (2010) 318:2-9.

In certain embodiments, the condition associated with angiogenesis isatherosclerosis. In certain embodiments, provided is a method oftreating atherosclerosis comprising administering to a subject in needthereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof. Evidence suggests that new angiogenesis occurs inatherosclerotic lesions, contributing to their growth and rupture.Therefore, inhibition of angiogenesis may be a therapeutic strategy forrestricting the expansion, growth, and ultimate rupture ofatherosclerotic plaques to prevent and treat atherosclerosis. See, e.g.,Ho-Tin-Noé et al., Trends Cariovasc. Med. (2011) 21:183-187.

In certain embodiments, the condition associated with angiogenesis is aproliferative disorder. In certain embodiments, provided is a method oftreating a proliferative disorder comprising administering to a subjectin need thereof a compound of Formula (A), (B), (C), (D), or (E) or apharmaceutically acceptable salt, quaternary amine, or N-oxide thereof.

Exemplary proliferative disorders include, but are not limited to,tumors (e.g., solid tumors), benign neoplasms, pre-malignant neoplasms(carcinoma in situ), and malignant neoplasms (cancers).

Exemplary cancers include, but are not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing's sarcoma, eye cancer (e.g., intraocular melanoma,retinoblastoma), familiar hypereosinophilia, gall bladder cancer,gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromaltumor (GIST), head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC),throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL)—also known as acutelymphoblastic leukemia or acute lymphoid leukemia (e.g., B-cell ALL,T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cellAML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML),and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL);lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) andnon-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large celllymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)),follicular lymphoma, chronic lymphocytic leukemia/small lymphocyticlymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-celllymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas,nodal marginal zone B-cell lymphoma, splenic marginal zone B-celllymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma,lymphoplasmacytic lymphoma (i.e., “Waldenström's macroglobulinemia”),hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma and primary central nervous system (CNS)lymphoma; and T-cell NHL such as precursor T-lymphoblasticlymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneousT-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome),angioimmunoblastic T-cell lymphoma, extranodal natural killer T-celllymphoma, enteropathy type T-cell lymphoma, subcutaneouspanniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); amixture of one or more leukemia/lymphoma as described above; andmultiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease,gamma chain disease, mu chain disease), hemangioblastoma, inflammatorymyofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g.,nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer(e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer(e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-smallcell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma(LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplasticsyndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g.,polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloidmetaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathicmyelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilicleukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma,neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2,schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreaticneuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovariancancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g.,pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm(IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of thepenis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT),prostate cancer (e.g., prostate adenocarcinoma), rectal cancer,rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamouscell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cellcarcinoma (BCC)), small bowel cancer (e.g., appendix cancer), softtissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma,malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma,fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat glandcarcinoma, synovioma, testicular cancer (e.g., seminoma, testicularembryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of thethyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer),urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's diseaseof the vulva).

In certain embodiments, the cancer is associated with CDK8 and/or CDK19kinase activity.

In certain embodiments, the cancer is a hematopoietic cancer. In certainembodiments, the hematopoietic cancer is a lymphoma. In certainembodiments, the hematopoietic cancer is a leukemia. In certainembodiments, the leukemia is acute myelocytic leukemia (AML). As shownin FIGS. 4, 5, 6, and 8, cortistatin A or cortistatin A analogs inhibitproliferation of AML cell lines in vitro and as shown in FIG. 12,cortistatin A inhibits AML progression in vivo.

In certain embodiments, the proliferative disorder is amyeloproliferative neoplasm. In certain embodiments, themyeloproliferative neoplasm (MPN) is primary myelofibrosis (PMF). Asshown in FIG. 8, cortistatin A inhibits the proliferation of cell linesderived from patients with MPNs and as shown in FIG. 14, cortistatin Ais efficacious in an in vivo model of PMF.

In certain embodiments, the cancer is a solid tumor. A solid tumor, asused herein, refers to an abnormal mass of tissue that usually does notcontain cysts or liquid areas. Different types of solid tumors are namedfor the type of cells that form them. Examples of classes of solidtumors include, but are not limited to, sarcomas, carcinomas, andlymphomas, as described above herein. Additional examples of solidtumors include, but are not limited to, squamous cell carcinoma, coloncancer, breast cancer, prostate cancer, lung cancer, liver cancer,pancreatic cancer, and melanoma.

Compounds of Formula (A), (B), (C), (D), or (E) and pharmaceuticallyacceptable salts, quaternary amines, and N-oxides thereof, may beformulated in dosage unit form for ease of administration and uniformityof dosage. It will be understood, however, that the total daily usage ofthe compositions comprising a compound as described herein will bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective dose level for anyparticular subject or organism will depend upon a variety of factorsincluding the disease, disorder, or condition being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

The compounds and compositions provided herein can be administered byany route, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),the condition of the subject (e.g., whether the subject is able totolerate oral administration).

The exact amount of a compound required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound(s), mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 0.1 mg to about10 mg, or about 0.1 mg to about 15 mg, of a compound per unit dosageform.

In certain embodiments, the compound may be administered orally orparenterally to an adult human at dosage levels sufficient to deliverfrom about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg,preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kgto about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and morepreferably from about 0.01 mg/kg to about 1 mg/kg, of subject bodyweight per day, one or more times a day, to obtain the desiredtherapeutic effect.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a compound or composition, as describedherein, can be administered in combination with one or more additionaltherapeutically active agents. The compounds or compositions can beadministered in combination with additional therapeutically activeagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body. It will also be appreciated that the therapy employedmay achieve a desired effect for the same disorder (for example, acompound can be administered in combination with an anti-inflammatoryagent, anti-cancer agent, etc.), and/or it may achieve different effects(e.g., control of adverse side-effects, e.g., emesis controlled by ananti-emetic).

The compound or composition can be administered concurrently with, priorto, or subsequent to, one or more additional therapeutically activeagents. In general, each agent will be administered at a dose and/or ona time schedule determined for that agent. In will further beappreciated that the additional therapeutically active agent utilized inthis combination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof the inventive compound with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved. In general,it is expected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

Exemplary additional therapeutically active agents include, but are notlimited to, small organic molecules such as drug compounds (e.g.,compounds approved by the Food and Drugs Administration as provided inthe Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins and cells. Incertain embodiments, the additional therapeutically active agent is ananti-cancer agent, e.g., radiation therapy and/or one or morechemotherapeutic agents.

Methods of Preparation

Still further provided are methods of preparing compounds of Formula(A), (B), (C), (D), and (E). An exemplary synthesis of compoundscontemplated herein is provided in Schemes 2 to 14.

The synthesis initially is contemplated using a compound of Formula (I)as starting material. Oxidation (e.g., DDQ, MnO₂) of estrone (wherein R³is —CH₃) or norestrone (wherein R³ is H) (I) provides the compound ofFormula (III). See, e.g., Stephan et al., Steroid, 1995, 60, 809-811.The compound of Formula (III) is protected as an acetal or ketal (e.g.,via reaction with HX^(A)R^(A), or HX^(A)R^(A)—R^(A)X^(A)H, wherein thetwo R^(A) groups are joined, wherein R^(B1) and R^(B2) are eachindependently —X^(A)R^(A)) to give a mixture (e.g., 1:1 mixture) of(IV)-A and (IV)-B. Exemplary conditions contemplated for protectioninclude PTSA and ethylene glycol, PTSA and CH(OMe)₃, PTSA and CH(OEt)₃,PTSA and 2,2-dimethyl-1,3-propandiol). The protected compounds are thenalkylated (e.g., methylated) using an alkylating agent (e.g., Me₂SO₄ andK₂CO₃, EtN(i-Pr)₂ and TMS-diazomethane) to afford (V)-A and (V)-B,wherein E is optionally substituted alkyl. See Scheme 2.

Scheme 3 provides other exemplary routes to provide a compound ofFormula (IV-B), e.g., wherein R³ is —CH₃. For example, the compound ofFormula (V)-B is achieved as racemic mixtures from 6-methoxy-1-tetralonein four steps as described in Scheme 3(A). For the Grignard reaction,see, e.g., Saraber et al., Tetrahedron, 2006, 62, 1726-1742. Forhydrogenation, see, e.g., Sugahara et al., Tetrahedron Lett, 1996, 37,7403-7406. Scheme 3(B) shows method to obtain enantiopure Torgov'sintermediate by chiral resolution. See, e.g., Bucourt et al., J. Bull.Soc. Chim. Fr. (1967) 561-563. Scheme 3(C) provides another method ofpreparing enantiopure Torgov's intermediate aided by enzymaticreduction. See, e.g., Gibian et al., Tetrahedron Lett. (1966)7:2321-2330.

With compounds of Formula (IV-A) and (IV-B) in hand, epoxidation/epoxideopening/epoxidation reactions are conducted (e.g., MMPP, mCPBA) inone-pot to provide the compound of Formula (IX-A) and (IX-B), which areunder equilibrium with (IX-A) as a major compound. See Schemes 4A and4B.

The compound of Formula (IX-A) and (IX-B) are exposed to Birch reductioncondition (e.g., Li/NH₃ and t-BuOH, Na/NH₃ and t-BuOH) to givedearomatized compound (X). C3 of A-ring is then protected as an acetalor ketal (e.g., via reaction with HX^(A)R^(A), orHX^(A)R^(A)—R^(A)X^(A)H, wherein the two R^(A) groups are joined, andwherein R^(B1) and R^(B2) are each independently —X^(A)R^(A)) to affordthe compound (XI). Exemplary protection conditions include PTSA andethylene glycol, PTSA and CH(OMe)₃, PTSA and CH(OEt)₃, and PTSA and2,2-dimethyl-1,3-propandiol. See Scheme 5.

The compound (XI) is converted to a compound of Formula (XIII) throughetherification (e.g., NBS, NIS, e.g., wherein X is Br or I). Thiscompound is then oxidized (e.g., SO₃.Py/DMSO and triethylamine, IBX,(COCl)₂/DMSO and triethylamine) to provide the compound of Formula(XIV). This compound is then treated with base (e.g., DBU,triethylamine) to provide the compound of Formula (XV). This compound isthen reduced (e.g., NaBH₄ and CeCl₃, L-selectride) to provide thecompound of Formula (XVI). See Scheme 6.

The compound of Formula (XVI) is then treated with cyclopropanationreagents (e.g., ZnEt₂ and ClCH₂I, ZnEt₂ and CH₂I₂, Zn—Cu and CH₂I₂) toprovide a compound of Formula (XVII). The alcohol of the cyclopropanatedproduct is activated, wherein LG¹ is a sulfonyl (e.g., the alcohol istreated with Tf₂O, MsCl, to provide an activated alcohol wherein LG¹ isTf or Ms) and treated with base (e.g., 2,6-di-t-butyl-4-methylpyridine,2,6-lutidine, triethylamine) to provide the compound of Formula (XX).See, e.g., Magnus et al., Org. Lett. 2009, 11, 3938-3941. See Scheme 7.

Protecting group on D-ring of the compound of Formula (XX) is thendeprotected under acidic conditions (e.g., PTSA and acetone/water,TFA/water) to provide the ketone intermediate of Formula (XXI). Thisproduct is treated with a compound of Formula R^(B1)-M (e.g.,R^(B1)—CeCl₂, R^(B1)—Mg) which is prepared from R^(B1)—X (e.g.,R^(B1)—Br, R^(B1)—I) to provide a compound of Formula (XXII), whereinR^(B1) is a non-hydrogen group as defined herein. The compound ofFormula (XXII) is activated (e.g., TFAA and pyridine, PhNCS and KH) toprovide a compound of Formula (XXIII). Reduction of the compound ofFormula (XXIII) (e.g., AIBN and Bu₃SnH) provides the compound of Formula(XXIV). For steps S14, S15 and S16, see, e.g., Flyer et al., Nature.Chem. 2010, 2, 886-892., and Yamashita et al., J. Org. Chem. 2011, 76,2408-2425. See Scheme 8A.

Compound (XXIV) may also be prepared from (XX) through conversion to anactivated alcohol, wherein LG² is a sulfonyl (e.g., the alcohol istreated with Tf₂O, MsCl, to provide an activated alcohol wherein LG² isTf or Ms; by triflation, e.g., KHMDS and PhNTf₂, LiHMDS and PhNTf₂, Tf₂Oand 2,6-di-t-butyl-4-methylpyridine) followed by palladium-catalyzedcross coupling with R^(B1)-M, wherein M is a substituted boron (e.g.,such as —B(R′)₂, wherein each R′ is —OR″ or alkyl wherein the alkyl andR″ is alkyl or may be joined to form a ring) to provide the compound ofFormula (XXVI). Exemplary palladium-catalyzed cross coupling conditionsinclude, but are not limited to, R^(B1)—B(pin), R^(B1)-(9-BBN—H),R^(B1)—OBBD, or R^(B1)—B(cat), and Pd(PPh₃)₄ and Na₂CO₃, or Pd(dppf)Cl₂and K₃PO₄) (pin=pinacol; cat=catechol;OBBD=9-oxa-10-brabicyclo[3.3.2]decane;9-BBN—H=9-broabicyclo[3.3.1]nonane). See, e.g., Nicolaou et al., J. Am.Chem. Soc. 2009, 131, 10587-10597. Hydrogenation of C16-C17 double bond(e.g., Pd/C and H₂, Raney Ni and H₂) gives the compound of Formula(XXIV). See Scheme 8B.

Any one of the compounds of Formula (XXVI) or (XXIV) may then bedeprotected (e.g., PTSA and acetone/water, TFA/water, HCl) and theresulting alpha carbon may be alkylated with an electrophile (e.g.,R⁵-LG, wherein LG is a leaving group, e.g., —OSO₂R^(aa)) to provide thecorresponding alkylated products, wherein R⁵ is a non-hydrogen group.See Scheme 9A and 9B.

The ketone compounds as provided in Scheme 9(A) and 9(B) can then betreated with an amine of formula H₂NR¹ to form the condensationproducts, imines, as depicted in Step S22. The ketone compounds can alsobe treated with an amine of formula HNR¹R², or salt thereof, underreductive amination conditions to provide the aminated products, asdepicted in Step S23. Exemplary reductive amination conditions include,but are not limited to, NaCNBH₃, NaCN(9BBN)H, or NaBH(OAc)₃ under acidicpH (e.g., pH of 3). The aminated products can further be oxidized to thecorresponding N-oxide, as depicted in Step S25. Exemplary oxidizingconditions include, but are not limited to, H₂O₂, mCPBA, or DMDO. SeeSchemes 10A to 10D.

The compound of Formula (XXV) can be converted to the compound ofFormula (XXV-i) through palladium-catalyzed carbonylative amination withCO and HN(R^(L))R^(B3) (e.g., Pd(PPh₃)₄ and triethylamine, Pd(dppf)Cl₂and triethylamine). Conditions for the following steps to get to thecompound of Formula (XXV-i), (XXV-iii), (XXV-iv), and (XXV-v) are thesame as described previously. See Scheme 11.

The ketone compounds as provided in Scheme 11 can then be converted tothe corresponding imines, amines, and N-oxides, as described previously.See Scheme 12A and 12B.

The monoketone compound (XXI) can be reductively aminated withHNR^(B4)R^(B5) (e.g., 1,2,3,4-tetrahydro-[2,7]naphthyridine) underconditions previously described to provide the compound of Formula(XXVII). Compound (XXVII) can be converted to the corresponding imines,amines, and N-oxides, as described previously. See Scheme 13.

The ketone may be further synthetically manipulated to provide othercompounds of interest. Taking the ketone of formula (XXIV-i) as anexample, the ketone may be related (as depicted in step S26) in thepresence of a reducing agent to provide the C-3 hydroxylated compound.See Scheme 14. Exemplary reducing agents include L-selectride,K-selectride, diisobutylaluminum hydride (DIBALH), and lithium aluminumhydride (LAH). Furthermore, various reducing agents will preferentiallygenerate one C-3 hydroxylated compounds as the major isomer over theother, e.g., using L-selectride the beta isomer is preferably generatedas the major isomer, while using lithium aluminum hydride (LAH) thealpha is preferably generated as the major isomer.

As used herein, a “major isomer” refers to the isomer that is producedin excess of the other isomer, i.e., greater than 50% of the sum of thetwo isomers produced from the reaction, e.g., greater than 60%, 70%,80%, 90%, or 95% of the sum of the two isomers produced from thereaction.

The C-3 hydroxylated compound of Formula (D) may then be activated to acompound of Formula (E) (e.g., by reaction with a group LG-C(═O)R^(A),wherein LG is a leaving group, either prior to commencing the reactionor in situ (during the reaction) via substitution with a group offormula —C(═O)R^(A) under Mitsunobu reaction conditions (e.g., withHOC(═O)R^(A), diethylazodicarboxylate (DEAD) or diisopropylazodicarboxylate (DIAD), and PPh₃)) and then treated with an amine offormula NHR¹R² to provide a compound of Formula (A) with inverted C3stereochemistry as the major isomer (as depicted in step S28).Alternatively, the C-3 hydroxylated compound of Formula (D) may betreated with base and a compound of formula R^(O)-LG, wherein LG is aleaving group, to provide a protected C3-hydroxyl compound withretention of C3-stereochemistry as the major isomer (as depicted in stepS27).

Thus, in one aspect, provided is a method of preparing a compound ofFormula (A):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof;the method comprising contacting a compound of Formula (B):

or a pharmaceutically acceptable salt thereof, provided R^(B1) andR^(B2) are not joined to form an oxo group; with an amine of formulaHNR¹R², or salt thereof, under reductive amination conditions. Incertain embodiments, the method comprises preparing one C3 isomer as themajor isomer over the other isomer. For example, in certain embodiments,the method comprises preparing the compound of Formula (A-1) as themajor isomer. In other embodiments, the method comprises preparing thecompound of Formula (A-2) as the major isomer.

In certain embodiments, the method further comprises oxiding thecompound of Formula (A) to provide an N-oxide of Formula (A-NO):

or a pharmaceutically acceptable salt thereof.

In other embodiments, provided is a method of preparing a compound ofFormula (D):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof;the method comprising contacting a compound of Formula (B):

or a pharmaceutically acceptable salt thereof, with a reducing agent. Incertain embodiments, the method comprises preparing one C3 isomer as themajor isomer over the other isomer. For example, in certain embodiments,the method comprises preparing the compound of Formula (D-1) as themajor isomer. In other embodiments, the method comprises preparing thecompound of Formula (D-2) as the major isomer.

In another aspect, provided is a method of preparing a compound ofFormula (E):

or a pharmaceutically acceptable salt thereof; the method comprisingcontacting a compound of Formula (D):

or a pharmaceutically acceptable salt thereof, with a compound offormula R^(O)-LG, wherein LG is a leaving group, to provide a compoundof Formula (E).

In another aspect, provided is a method of preparing a compound ofFormula (A):

or a pharmaceutically acceptable salt, quaternary amine salt, or N-oxidethereof; the method comprising providing a compound of Formula (E):

wherein R^(O) is —C(═O)R^(A), or a pharmaceutically acceptable saltthereof; and treating the compound of Formula (E) with a compound offormula NHR¹R², to provide a compound of Formula (A). In certainembodiments, the compound of Formula (E) wherein R^(O) is —C(═O)R^(A) isgenerated in situ from the activation of a compound of Formula (D) witha compound of formula R^(O)-LG. In certain embodiments, the compound ofFormula (E) is a compound of Formula (E-1), and the method generates acompound of Formula (A-2) as the major isomer. In certain embodiments,the compound of Formula (E) is a compound of Formula (E-2), and themethod generates a compound of Formula (A-1) as the major isomer.

Mutants and Assay Methods

As generally described herein, further provided novel CDK8 and CDK19point mutants, and methods of their use.

CDK8 and CDK19 with point mutations at Trp105, such as Trp105Met, mayretain cellular and in vitro activity but be insensitive to inhibitionby cortistatins or cortistatin analogs. As shown in FIG. 10, FLAG-CDK8W105M and FLAG-CDK19 W105M expression reduces the sensitivity of MOLM-14cells to growth inhibition by cortistatin A and FLAG-CDK8W105M retainskinase activity in the presence of cortistatin A. CDK8 and/or CDK19Trp105 point mutants may be used together with a cortistatin orcortistatin analog to validate CDK8 and CDK19 kinase activity indifferent contexts, including as drug targets for different diseases. Inaddition, CDK8 and/or CDK19 Trp105 point mutants may be used togetherwith a cortistatin or cortistatin analog to elucidate CDK8 and/or CDK19kinase substrates and signaling pathways. These point mutants may beexpressed in cells by means of overexpression or knock-in and may beconstitutively expressed or inducibly expressed. These point mutants mayalso be used in screening programs to discover new chemical structuresthat selectively target CDK8 and/or CDK19.

The wild-type amino acid sequence for CDK8 is provided below and in SEQID NO: 1. Trp105 is underlined. The CDK8 Trp105 point mutant usefulherein comprises an amino acid substitution at position 105.

MDYDFKVKLSSERERVEDLFEYEGCKVGRGTYGHVYKAKRKDGKDDKDYALKQIEGTGISMSACREIALLRELKHPNVISLQKVFLSHADRKVWLLFDYA EHDL WHIIKFHRASKANKKPVQLPRGMVKSLLYQILDGIHYLHANWVLHRDLKPANILVMGEGPERGRVKIADMGFARLFNSPLKPLADLDPVVVTFWYRAPELLLGARHYTKAIDIWAIGCIFAELLTSEPIFHCRQEDIKTSNPYHHDQLDRIFNVMGFPADKDWEDIKKMPEHSTLMKDFRRNTYTNCSLIKYMEKHKVKPDSKAFHLLQKLLTMDPIKRITSEQAMQDPYFLEDPLPTSDVFAGCQIPYPKREFLTEEEPDDKGDKKNQQQQQGNNHTNGTGHPGNQDSSHTQGPPLKKVRVVPPTTTSGGLIMTSDYQRSNPHAAYPNPGPSTSQPQSSMGYSAT SQQPPQYSHQTHRY

The wild-type amino acid sequence for CDK19 is provided below and in SEQID NO: 2. Trp105 is underlined. The CDK19 Trp105 point mutant usefulherein comprises an amino acid substitution at position 105.

MDYDFKAKLAAERERVEDLFEYEGCKVGRGTYGHVYKARRKDGKDEKEYALKQIEGTGISMSACREIALLRELKHPNVIALQKVFLSHSDRKVWLLFDYA EHDL WHIIKFHRASKANKKPMQLPRSMVKSLLYQILDGIHYLHANWVLHRDLKPANILVMGEGPERGRVKIADMGFARLFNSPLKPLADLDPVVVTFWYRAPELLLGARHYTKAIDIWAIGCIFAELLTSEPIFHCRQEDIKTSNPFHHDQLDRIFSVMGFPADKDWEDIRKMPEYPTLQKDFRRTTYANSSLIKYMEKHKVKPDSKVFLLLQKLLTMDPTKRITSEQALQDPYFQEDPLPTLDVFAGCQIPYPKREFLNEDDPEEKGDKNQQQQQNQHQQPTAPPQQAAAPPQAPPPQQNSTQTNGTAGGAGAGVGGTGAGLQHSQDSSLNQVPPNKKPRLGPSGANSGGPVMPSDYQHSSSRLNYQSSVQGSSQSQSTLGYSSSSQQSSQYHPSHQAH RY

Thus, in one aspect, provided is a method of validating CDK8 and/orCDK19 kinase activity in a cell by contacting a CDK8 or CDK19 Trp105point mutant and a cortistatin or cortistatin analog, as describedherein. In another aspect, provided is a method of validating CDK8and/or CDK19 kinase activity in a cell by expressing CDK8 or CDK19Trp105 point mutant in a cell to desensitize the cell to cortistatins orcortistatin analog, as described herein.

In another aspect, provided is a CDK8 Trp105 point mutant. In certainembodiments, the CDK8 Trp105 point mutant has an amino acid sequencethat a degree of homology to the amino acid sequence of SEQ ID NO: 1 ofat least about 80%, e.g., at least about 85%, at least about 90%, atleast about 95%, or at least about 97%. Further provided is a proteinthat has a degree of homology to the amino acid sequence of SEQ ID NO: 1of at least about 80%, e.g., at least about 85%, at least about 90%, atleast about 95%, or at least about 97%.

In yet another aspect, provided is a CDK19 Trp105 point mutant. Incertain embodiments, the CDK19 Trp105 point mutant has an amino acidsequence that a degree of homology to the amino acid sequence of SEQ IDNO: 2 of at least about 80%, e.g., at least about 85%, at least about90%, at least about 95%, or at least about 97%. Further provided is aprotein that has a degree of homology to the amino acid sequence of SEQID NO: 2 of at least about 80%, e.g., at least about 85%, at least about90%, at least about 95%, or at least about 97%.

In yet another aspect, provided is a CDK19 Trp105 point mutant. Incertain embodiments, the CDK19 Trp105 point mutant has an amino acidsequence as recited in SEQ ID NO. 2. Further provided is a protein thatis 80% homologous to SEQ ID NO. 2.

In one embodiment, the CDK8 Trp105 point mutant useful in the methodsherein has a methionine substitution located at position 105 of the CDK8polypeptide of SEQ ID NO: 1. In one embodiment, the CDK19 Trp105 pointmutant useful in the methods herein has a methionine substitutionlocated at position 105 of the CDK19 polypeptide of SEQ ID NO: 2. In anyof the foregoing embodiments, another amino acid may be substituted inplace of Trp105 such as other natural or non-natural amino acids.Non-limiting examples of natural amino acids include isoleucine,leucine, valine, alanine, lysine, arginine, threonine, glutamic acid,methionine, and cysteine. Non-limiting examples of non-natural aminoacids include ethionine or norleucine.

In one embodiment, the CDK8 Trp105 point mutant useful herein have anamino acid sequence which has a degree of homology to the amino acidsequence of SEQ ID NO: 1 of at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 97%. In anembodiment, the CDK8 Trp105 point mutant of SEQ ID NO: 1 comprisesadditional one or more substitutions, deletions, and/or insertions ofone or more amino acids. In another embodiment, the CDK8 Trp105 pointmutant is an allelic variant of SEQ ID NO: 1. An allelic variant denotesany of two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene.

In one embodiment, the CDK8 Trp105 point mutant is a fragment of SEQ IDNO: 1, or an allelic variant thereof, having CDK8 activity. A fragmentof SEQ ID NO: 1 is a CDK8 Trp105 point mutant having one or more aminoacids deleted from the amino and/or carboxyl terminus of SEQ ID NO: 1,or an allelic variant thereof.

The CDK8 Trp105 point mutant useful herein have at least about 20%, atleast about 40%, at least about 60%, at least about 80%, at least about90%, or at least about 95% of the CDK8 kinase activity of the CDK8polypeptide of SEQ ID NO: 1.

In one embodiment, the CDK19 Trp105 point mutant useful herein have anamino acid sequence which has a degree of homology to the amino acidsequence of SEQ ID NO: 2 of at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 97%. In anembodiment, the CDK19 Trp105 point mutant of SEQ ID NO: 2 comprisesadditional one or more substitutions, deletions, and/or insertions ofone or more amino acids. In another embodiment, the CDK19 Trp105 pointmutant is an allelic variant of SEQ ID NO: 2.

In one embodiment, the CDK19 Trp105 point mutant is a fragment of SEQ IDNO:2, or an allelic variant thereof, having CDK19 activity. A fragmentof SEQ ID NO:2 is a CDK19 Trp105 point mutant having one or more aminoacids deleted from the amino and/or carboxyl terminus of SEQ ID NO:2, oran allelic variant thereof.

The CDK19 Trp105 point mutant useful herein have at least about 20%, atleast about 40%, at least about 60%, at least about 80%, at least about90%, or at least about 95% of the CDK19 kinase activity of the CDK19polypeptide of SEQ ID NO:2.

In one embodiment, the CDK8 or CDK19 Trp105 point mutant useful hereincomprises a tag such as a peptide tag (e.g., a FLAG® tag, GST tag, humaninfluenza hemagglutinin (HA) tag, chitin binding protein (CBP) tag,maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag,poly(His) tag, V5-tag, Myc-tag) or fluorescent tags (e.g., eGFP tag,mCherry tag).

Also contemplated is a method of predicting or determining a subject'sresponse to cortistatin or cortistatin analogs. For example, provided isa method of analyzing a sample from a subject to determine if a subjecthas a point mutation located at Trp105 of CDK8 and/or CDK19. In oneembodiment the point mutation is a Trp105Met. The presence of one ormore of Trp105 mutations is generally indicative of a decreased orineffective response of a subject to the cortistatin or cortistatinanalogs. In one embodiment, a different drug besides cortistatin orcortistatin analogs is administered to the subject if the subject has apoint mutation located at Trp105 of CDK8 and/or CDK19.

Examples

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Synthetic Methods Materials and Instrumentation

All reactions were performed in flame-dried glassware under a positivepressure of argon unless otherwise noted. Flash column chromatographywas performed as described by Still et al., J. Org. Chem. 1978, 43,2923-2925 employing silica gel 60 (40-63 μm, Whatman).

Commercial reagents and solvents were used as received with thefollowing exceptions: tetrahydrofuran (THF), dichloromethane (CH₂Cl₂)were degassed with argon and passed through a solvent purificationsystem (designed by J. C. Meyer of Glass Contour) utilizing aluminacolumns as described by Pangborn et al., Organometallics 1996, 15,1518-1520. Pyridine and triethylamine were distilled over calciumhydride before use. The celite used was Celite® 545, purchased from J.T.Baker. The molarities of n-butyllithium solutions were determined bytitration using 1,10-phenanthroline as an indicator (average of threedeterminations).

¹H NMR spectra were recorded with a Varian INOVA-600 or Varian INOVA-500spectrometer. Proton chemical shifts are reported in parts per million(δ scale) and are calibrated using residual undeuterated solvent as aninternal reference (CDCl₃: δ 7.26 (CHCl₃), C₆D₆: δ 7.15 (C₆D₅H)). Datafor ¹H NMR spectra are reported as follows: chemical shift (δ ppm)(multiplicity, coupling constant (Hz), integration). Multiplicities arereported as follows: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet, br=broad, app=apparent, or combinations thereof. ¹³C NMRspectra were recorded with a Varian INOVA-500 spectrometer.High-resolution mass spectra (HRMS) were obtained from the HarvardUniversity Mass Spectrometry Laboratory where electrospray ionization(ESI) mass spectroscopy (MS) experiments were performed on an Agilent6210 TOF LC/MS instrument.

Example 1 Synthesis of Isoquinoline-Containing Compounds

Route 1 Synthesis of 8,9-unsaturated methoxyethyleneketone from6-methoxy-1-tetralone (compound 1)

The Grignard reaction was done with 20.0 g (113 mmol, 1.00 equiv) of6-methoxy-1-tetralone and the product was used without purification byflash chromatography. See, e.g., Saraber et al., Tetrahedron 2006, 62,1726-1742. To a solution of Grignard reaction product and2-methyl-1,3-pentadienone (12.8 g, 114 mmol, 1.01 equiv) in xylene (140mL) was added AcOH (64.6 mL, 1.13 mol, 10.0 equiv) and the reactionmixture was warmed to reflux. After 2 h, the reaction was allowed tocool to room temperature and the concentrated under reduced pressure.The mixture of 1:1 of toluene and ethyl ether was added to dissolve thesolid residue and the mixture was filtered. The filtrate was washedsequentially with saturated NaHCO₃ solution (200 mL) and brine, driedover MgSO₄, and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, eluent: 20:1:1Hexanes:EtOAc:DCM) to afford the Torgov's diene. Spectral data wasconsistent with those previously reported. See, e.g., Soorukram, D.;Knochel, P. Org. Lett. 2007, 9, 1021-1023. The Torgov's diene wasconverted to 8,9-unsaturated methoxyethyleneketone compound 1 (15.0 g,47% over 3 steps) based on the literature known procedure. See, e.g.,Sugahara et al., Tetrahedron Lett. 1996, 37, 7403-7406.

Route 1 Synthesis of 8,9-Unsaturated Methoxyethyleneketal (Compound 2)

To a solution of compound 1 (15.0 g, 53.1 mmol, 1.0 equiv) in benzene(215 mL) and ethylene glycol (72 mL) was added oxalic acid (2.30 g, 12.1mmol, 0.22 equiv). The reaction mixture was allowed to warm to refluxand water was trapped by Dean-Stark apparatus. After 16 h, the reactionwas cool to room temperature and saturated NaHCO₃ solution (150 mL) wasadded. The organic and aqueous layers were separated and the aqueousphase was extracted with ethyl acetate (2×200 mL). The combined organicphases were washed with brine (150 mL) and dried over Na₂SO₄. Thesolvent was evaporated under reduced pressure and the residue waspurified by flash chromatography (silica gel, eluent: 15:1Hexanes:EtOAc) to provide 8,9-unsaturated methoxyethyleneketal compound2 (15.5 g, 89%). ¹H NMR (500 MHz, CDCl₃) Shift=7.13 (d, J=8.3 Hz, 1H),6.73-6.67 (m, 2H), 4.05-3.85 (m, 4H), 3.79 (s, 3H), 2.82-2.65 (m, 2H),2.52-2.45 (m, 2H), 2.23-2.17 (m, 2H), 2.14 (ddd, J=2.2, 11.6, 14.0 Hz,1H), 1.99-1.82 (m, 4H), 1.64 (td, J=4.2, 12.2 Hz, 1H), 1.49 (dq, J=6.8,11.6 Hz, 1H), 0.86 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₁H₂₇O₃ [M+H]⁺:327.1955. found 327.1947.

Route 1 Synthesis of Epoxy Alcohols 3 and 3a

A solution of 8,9-unsaturated ethyleneketal 2 (1.63 g, 5.00 mmol, 1.0equiv) in CHCl₃ (50 mL) was cooled to 0° C. and mCPBA (77% max, 2.46 g,11.0 mmol, 2.2 equiv) was added. The reaction mixture was stirred for 10min at 0° C. and warmed to room temperature. After additional 50 min,10% Na₂S₂O₃ solution (40 mL) and saturated NaHCO₃ solution (40 mL) weresequentially added. The organic and aqueous layers were separated andthe aqueous phase was extracted with dichloromethane (3×50 mL). Thecombined organic phases were washed with brine (50 mL), dried overNa₂SO₄, and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, eluent: 3:1→1:1Hexanes:EtOAc) to afford epoxy alcohol 3 and 3a (1.40 g, 75%). 3 and 3awere under equilibration in any solvent, with a major of 3. H NMR wasanalyzed for epoxy alcohol 3. Where indicted, cortistatin analogs (12,13, 14A, 14B, 15B, 16B, and 17B) were applied to the biologicalexperiments as racemic mixtures constructed from 6-methoxy-1-tetralone.

¹H NMR (500 MHz, CDCl₃) Shift=7.77 (d, J=8.3 Hz, 1H), 6.76 (dd, J=2.0,8.3 Hz, 1H), 6.63 (d, J=2.0 Hz, 1H), 4.78 (dd, J=7.8, 9.8 Hz, 1H),3.95-3.87 (m, 4H), 3.78 (s, 3H), 2.84 (dt, J=5.9, 14.4 Hz, 1H), 2.49(dd, J=4.4, 15.1 Hz, 1H), 2.36-2.29 (m, 1H), 2.26 (dd, J=5.9, 14.2 Hz,2H), 2.06 (t, J=11.7 Hz, 1H), 1.97 (dd, J=7.3, 12.2 Hz, 1H), 1.94-1.88(m, 2H), 1.75 (dt, J=5.4, 14.2 Hz, 1H), 1.63-1.53 (m, 1H), 1.46 (t,J=11.0 Hz, 1H), 0.75 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₁H₂₇O₅[M+H]⁺: 359.1853. found 359.1852.

Route 2 Synthesis of 8,9 and 9,11-Unsaturated MethoxyethyleneketalCompounds 2 and 4

The DDQ oxidation was done with 22.0 g (81.4 mmol, 1.0 equiv) of estroneand the ^(product) was used without purification by flashchromatography. See, e.g., Stephan et al., Steroid. 1995, 60, 809-811.To a solution of 9,11-unsaturated estrone in benzene (375 mL) was addedethylene glycol (110 mL, 1.99 mol, 24.4 equiv) and PTSA (3.00 g, 16.3mmol, 0.20 equiv). The reaction mixture was warmed to reflux and waterwas trapped by Dean-Stark apparatus. After 18 h, the reaction wasallowed to cool to room temperature and saturated NaHCO₃ solution (300mL) was applied. The aqueous phase was extracted with ethyl acetate(2×300 mL) and the combined organic phases were washed with brine (200mL). The organic phase was dried (Na₂SO₄) and the solvent was evaporatedunder reduced pressure. The product was used in the next step withoutfurther purification.

The ethyleneketal (mixture of the 8,9 and 9,11-unsaturated regioisomers)was dissolved in acetone (420 mL) and K₂CO₃ (22.5 g, 163 mmol, 2.00equiv) was added. This was followed by the addition of Me₂SO₄ (9.30 mL,97.6 mmol, 1.20 equiv) and the reaction mixture was warmed to reflux.After 18 h, the reaction was allowed to cool to room temperature and theacetone was evaporated. 2M NaOH solution was added (300 mL) and theaqueous phase was extracted with ethyl acetate (2×300 mL). The combinedorganic phases were dried (Na₂SO₄) and the solvent was evaporated underreduced pressure. The residue was purified by flash chromatography(silica gel, eluent: 15:1 Hexanes:EtOAc) to afford mixture of 8,9 and9,11-unsaturated methoxyethyleneketal compounds 2 and 4 (16.3 g, 61% inthree steps, ˜4:5 mixture of 8,9-unsaturated:9,11-unsaturatedregioisomers).

For 9,11-unsaturated isomer, only distinguishable peaks were assigned:¹H NMR (500 MHz, CDCl₃) Shift=7.53 (d, J=8.8 Hz, 1H), 6.60 (d, J=2.0 Hz,1H), 6.13 (td, J=2.6, 5.0 Hz, 1H), 3.79 (s, 3H), 2.59 (td, J=3.2, 17.6Hz, 1H), 2.09-2.00 (m, 3H), 1.45-1.33 (m, 2H), 0.90 (s, 3H). HRMS (ESI)(m/z) calc'd for C₂₁H₂₇O₃ [M+H]⁺: 327.1955. found 327.1951.

Route 2 Epoxy Alcohol Compounds 3 and 3a

To a solution of mixture of 8,9 and 9,11-unsaturated ethyleneketalcompounds 2 and 4 (15.7 g, 48.1 mmol, 1.00 equiv) in dichloromethane(700 mL) was added magnesium monoperoxyphthalate hexahydrate (68.4 g,111 mmol, 2.30 equiv) and water (4.8 mL). The reaction mixture wasstirred for 20 h at room temperature and then quenched with the mixtureof 10% aqueous Na₂S₂O₃ (300 mL) and saturated NaHCO₃ solution (300 mL).The organic and aqueous layers were separated and the aqueous phase wasextracted with dichloromethane (2×500 mL). The combined organic phaseswere washed with brine (300 mL) and dried (Na₂SO₄). The solvent wasevaporated under reduced pressure and the residue was purified by flashchromatography (silica gel, eluent: 3:1→2:1 Hexanes:EtOAc) to provideepoxy alcohol 3 and 3a (8.60 g, 50%). Spectral data was consistent withepoxy alcohol 3 and 3a constructed from 8,9-unsaturatedmethoxyethyleneketal 2.

Synthesis of Diol Compound 5

Ammonia gas was condensed (240 mL) and to the liquid ammonia was addedLi (3.90 g, 565 mmol, 25.0 equiv) at −78° C. After stirring for 30 min,epoxy alcohol 3 and 3a (8.10 g, 22.6 mmol, 1.0 equiv) in THF (110 mL)was cannulated and stirred additional 1.5 h at that temperature. To thereaction mixture was added the mixture of t-BuOH (32 mL) and THF (16 mL)at −78° C. and stirred additional 20 min at that temperature. Themixture of t-BuOH (92 mL) and THF (38 mL) was added followed by benzene(50 mL) and water (50 mL) at −78° C., and the flask was opened to gentlyevaporate liquid ammonia by removing the cooling bath. Water (200 mL)was added and the aqueous phase was extracted with ethyl acetate (2×250mL). The combined organic phases were washed with brine (150 mL), dried(Na₂SO₄), and concentrated under reduced pressure. The product was usedin the next step without further purification.

To a solution of Birch reduction product in THF (300 mL) and ethyleneglycol (75 mL) was added PTSA (430 mg, 2.26 mmol, 0.10 equiv). Thereaction mixture was stirred for 30 min at room temperature andsaturated NaHCO₃ solution (200 mL) was added. The organic and aqueouslayers were separated and the aqueous phase was extracted with ethylacetate (4×250 mL). The combined organic phases were washed with brine(200 mL) and dried (Na₂SO₄). The solvent was evaporated under reducedpressure and the residue was purified by flash chromatography (silicagel, eluent: 4:1 Hexanes:EtOAc→100% EtOAc→10:1 EtOAc:MeOH) to providediol 5 (4.60 g, 52%).

¹H NMR (500 MHz, C₆D₆) Shift=3.67-3.42 (m, 9H), 3.25-3.14 (m, 1H), 2.40(dd, J=5.9, 13.2 Hz, 1H), 2.31 (br. s, 2H), 2.23-2.09 (m, 2H), 2.03 (t,J=10.7 Hz, 1H), 1.97-1.90 (m, 2H), 1.89 (dd, J=8.3, 14.2 Hz, 1H),1.85-1.75 (m, 4H), 1.66-1.50 (m, 4H), 1.00 (s, 3H). HRMS (ESI) (m/z)calc'd for C₂₂H₃₂NaO₆ [M+Na]⁺: 415.2091. found 415.2076.

Synthesis of Enone Compound 6

To a solution of diol 5 (4.05 g, 10.3 mmol, 1.00 equiv) indichloromethane (230 mL) was added NBS (2.00 g, 11.4 mmol, 1.10 equiv)at one portion at −10 OC and the reaction mixture was warmed to roomtemperature. The reaction was monitored by TLC (about 30 min for thecompletion). Once the reaction is done, the reaction mixture was cooledto −40° C. and triethylamine (17.3 mL, 124 mmol, 12.0 equiv) was added.Pre-stirred SO₃.Py (16.4 g, 103 mmol, 10.0 equiv) in DMSO (115 mL) for20 min at room temperature was added to the reaction mixture at −40° C.,which was subsequently allowed to warm slowly to −10° C. After 4 h,saturated NH₄Cl solution (130 mL) was added and the reaction was allowedto warm to room temperature. The organic and aqueous layers wereseparated and the aqueous phase was extracted with dichloromethane(2×200 mL). The combined organic phases were washed with brine (150 mL),dried over Na₂SO₄, and concentrated under reduced pressure. The productwas used without further purification.

The oxidation product was dissolved in dichloromethane (300 mL) and thereaction mixture was cooled to −40° C. followed by the slow addition ofDBU (3.90 mL, 25.6 mmol, 2.50 equiv). After 15 min, saturated NH₄Clsolution (130 mL) was added and the reaction was allowed to warm to roomtemperature. The organic and aqueous layers were separated and theaqueous phase was extracted with dichloromethane (2×200 mL). Thecombined organic phases were washed with brine (150 mL), dried overNa₂SO₄, and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, eluent: 3:1→1:1Hexanes:EtOAc) to afford enone 6 (3.16 g, 80% in three steps).

¹H NMR (500 MHz, C₆D₆) Shift=3.58-3.51 (m, 1H), 3.49-3.34 (m, 6H),3.28-3.23 (m, 2H), 3.19 (dt, J=4.2, 7.7 Hz, 1H), 2.80 (d, J=16.1 Hz,1H), 2.60 (ddd, J=6.8, 12.7, 19.0 Hz, 1H), 2.55 (d, J=13.2 Hz, 1H), 2.43(d, J=16.1 Hz, 1H), 2.31 (dd, J=1.5, 13.2 Hz, 1H), 1.98-1.88 (m, 2H),1.88-1.80 (m, 3H), 1.71 (ddd, J=4.2, 9.6, 11.6 Hz, 1H), 1.68-1.59 (m,3H), 1.20 (ddd, J=3.7, 8.4, 11.4 Hz, 1H), 0.90 (s, 3H). HRMS (ESI) (m/z)calc'd for C₂₂H₂₈NaO₆ [M+Na]⁺: 411.1778. found 411.1786.

Synthesis of Allylic Alcohol Compound 7

To a solution of enone 6 (3.20 g, 8.32 mmol, 1.00 equiv) in MeOH (150mL) and THF (20 mL) was added CeCl₃.7H₂O (9.20 g, 24.7 mmol, 3.00 equiv)at room temperature. After stirring 5 min, the reaction was cooled to−20° C. followed by the addition of NaBH₄ (623 mg, 16.5 mmol, 2.00equiv). After 30 min, saturated NH₄Cl solution (50 mL) and water (50 mL)was added, which was allowed to warm to room temperature. The aqueousphase was extracted with ethyl acetate (3×200 mL) and the combinedorganic phases were washed with brine (150 mL), dried over Na₂SO₄, andconcentrated under reduced pressure. The residue was purified by flashchromatography (silica gel, eluent: 20:1 DCM:MeOH) to afford allylicalcohol 7 (2.72 g, 85%).

¹H NMR (500 MHz, C₆D₆) Shift=4.39-4.30 (m, 1H), 3.58-3.36 (m, 8H), 3.22(dd, J=3.7, 16.4 Hz, 1H), 2.94 (dd, J=7.1, 12.5 Hz, 1H), 2.66 (d, J=13.2Hz, 1H), 2.49-2.41 (m, 1H), 2.39 (dd, J=2.2, 12.9 Hz, 1H), 2.07-1.99 (m,1H), 1.96-1.79 (m, 6H), 1.73 (br. s, 3H), 1.66-1.57 (m, 1H), 1.15-1.07(m, 1H), 0.86 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₂H₃₀NaO₆ [M+Na]⁺:413.1935. found 413.1942.

Synthesis of Cyclopropane Compound 8

To a solution of ClCH₂I (1.98 mL, 27.1 mmol, 4.00 equiv) in1,2-dichloroethane (140 mL) was added a solution of Et₂Zn in diethylether (1M, 13.6 mL, 13.6 mmol, 2.00 equiv) at 0° C. After stirring 5min, allylic alcohol 7 (2.65 g, 6.79 mmol, 1.00 equiv) in1,2-dichloroethane (70 mL) was added to the reaction flask at 0° C.After 30 min, the reaction was quenched by saturated NH₄Cl solution (100mL) and allowed to warm to room temperature. The organic and aqueouslayers were separated and the aqueous phase was extracted withdichloromethane (2×120 mL). The combined organic phases were washed withbrine (100 mL), dried over Na₂SO₄, and concentrated under reducedpressure. The residue was purified by flash chromatography (silica gel,eluent: 2:1→1:1 Hexanes:EtOAc) to afford cyclopropane 8 (2.59 g, 93%).

¹H NMR (500 MHz, C₆D₆) Shift=3.92 (dd, J=3.7, 11.0 Hz, 1H), 3.51-3.40(m, 8H), 2.72 (dd, J=7.1, 12.9 Hz, 1H), 2.39 (dd, J=5.4, 17.6 Hz, 1H),2.38 (d, J=12.2 Hz, 1H), 2.15 (d, J=12.2 Hz, 1H), 2.12 (dt, J=4.9, 12.2Hz, 1H), 2.02 (ddd, J=2.9, 11.2, 14.6 Hz, 1H), 1.92-1.82 (m, 3H),1.82-1.73 (m, 2H), 1.69-1.54 (m, 5H), 1.52 (dd, J=6.1, 12.0 Hz, 1H),1.49-1.44 (m, 1H), 0.98 (s, 3H), 0.86 (d, J=2.4 Hz, 1H), 0.15 (d, J=2.9Hz, 1H). HRMS (ESI) (m/z) calc'd for C₂₃H₃₂NaO₆ [M+Na]⁺: 427.2091. found427.2088.

Synthesis of Oxabicyclo[3.2.1]Octane Compound 9

Cyclopropane 8 (2.45 g, 6.06 mmol, 1.00 equiv) and2,6-di-tert-butyl-4-methylpyridine (4.40 g, 21.2 mmol, 3.50 equiv) wereazeotropically dried with benzene and dissolved in dichloromethane (120mL). 4 Å molecular sieves (3.1 g) were added and the reaction flask wascooled to 0° C. A solution of triflic anhydride in dichloromethane (1 M,12.1 mL, 12.1 mmol 2.00 equiv) was added dropwise and the ice bath wasremoved to warm the reaction flask to room temperature. After 2 h, thereaction was quenched with triethylamine (20 mL) and the filteredthrough a pad of celite. Saturated NaHCO₃ solution (100 mL) was addedand the aqueous phase was extracted with dichloromethane (2×120 mL). Thecombined organic phases were washed with brine (100 mL), dried overNa₂SO₄, and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, eluent: 3:1Pentane:Diethyl ether) to afford oxabicyclo[3.2.1]octane compound 9(1.42 g, 60%). See also Magnus et al., Org. Lett. 2009, 11, 3938-3941.

¹H NMR (500 MHz, CDCl₃) Shift=5.73 (s, 1H), 5.29-5.26 (m, 1H), 4.04-3.76(m, 8H), 2.58-2.50 (m, 1H), 2.46 (t, J=15.1 Hz, 1H), 2.31-2.24 (m, 2H),2.19 (t, J=11.2 Hz, 1H), 2.09 (d, J=13.2 Hz, 1H), 1.99 (dt, J=4.4, 13.2Hz, 1H), 1.94 (dd, J=2.4, 13.2 Hz, 1H), 1.91-1.84 (m, 1H), 1.83-1.71 (m,3H), 1.71-1.53 (m, 5H), 0.88 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₃H₃₀O₅ [M+H]⁺: 387.2166. found 387.2180.

Synthesis of Monoketone Compound 10

To a solution of bisethyleneketal 9 (110 mg, 285 μmol, 1.0 equiv) inacetone (14.6 mL) and water (3.6 mL) was added PTSA (21.6 mg, 85.2 μmol,0.30 equiv) and the reaction mixture was stirred for 3 d. SaturatedNaHCO₃ solution (5 mL) and ethyl acetate (25 mL) were sequentially addedto the reaction. The layers were separated and the aqueous layer wasextracted with ethyl acetate (2×15 mL). The organic layers werecombined, washed with brine (20 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The resulting residue was then purified by flashchromatography (silica gel, eluent: 4:1 Hexanes:EtOAc) to affordmonoketone 10 (79.0 mg, 81%).

¹H NMR (500 MHz, CDCl₃) Shift=5.73 (s, 1H), 5.29-5.25 (m, 1H), 3.98-3.90(m, 4H), 2.48 (dd, J=8.8, 19.5 Hz, 1H), 2.46-2.40 (m, 1H), 2.36 (dd,J=5.9, 12.7 Hz, 1H), 2.34-2.25 (m, 2H), 2.24-2.08 (m, 5H), 2.09 (d,J=13.2 Hz, 1H), 1.95 (dd, J=2.4, 13.2 Hz, 1H), 1.90-1.81 (m, 1H),1.79-1.70 (m, 2H), 1.70-1.61 (m, 2H), 0.89 (s, 3H). HRMS (ESI) (m/z)calc'd for C₂₁H₂₇O₄ [M+H]⁺: 343.1909. found 343.1919.

Synthesis of 1-Chloroisoquinoline Adduct Compound 11

CeCl₃ (565 mg, 2.30 mmol, 10.0 equiv) in reaction flask was heated at140° C. under vacuum for 2 h. The flask was charged with Ar and cooledto 0° C. After 30 min, THF (2.8 mL) was added and stirred at 0° C. for 2h. The flask was then allowed to warm to room temperature and stirredfor additional 16 h.

1-Chloro-7-iodoisoquinoline was synthesized following the procedureprovided in Subasinghe et al., Bioorg. Med. Chem. Lett. 2013, 23,1063-1069.

To a solution of CeCl₃/THF complex was added 1-chloro-7-iodoisoquinoline(396 mg, 1.40 mmol, 6.00 equiv) in THF (1.4 mL) followed by stirring for10 min at room temperature, which was then allowed to cool to −78° C. Asolution of n-butyllithium in hexanes (1.6 M, 716 μL, 1.10 mmol, 5.00equiv) was then added dropwise. The reaction mixture was stirredadditional 30 min at the same temperature and monoketone 10 (78.5 mg,229 μmol, 1.00 equiv) was cannulated in THF (1.4 mL). After additional30 min, saturated NH₄Cl solution (5 mL) was added to the stirredreaction mixture, which was then allowed to warm to room temperature.The mixture was diluted with EtOAc (5 mL) and the layers were separated.The aqueous layer was extracted with EtOAc (3×5 mL) and the organiclayers were combined, washed with brine (5 mL), and dried over Na₂SO₄and concentrated under reduced pressure. The resulting residue was thenpurified by flash chromatography (silica gel, eluent: 2:1 Hexanes:EtOAc)to provide 1-chloroisoquinoline adduct 11 (115 mg, 97%).

¹H NMR (500 MHz, CDCl₃) Shift=8.34 (br. s, 1H), 8.24 (d, J=5.9 Hz, 1H),7.89-7.83 (m, 1H), 7.76 (d, J=8.3 Hz, 1H), 7.56 (d, J=5.9 Hz, 1H), 5.63(s, 1H), 5.16-4.99 (m, 1H), 4.02-3.87 (m, 4H), 2.62 (ddd, J=4.4, 9.8,14.2 Hz, 1H), 2.48-2.38 (m, 2H), 2.36-2.26 (m, 3H), 2.26-2.19 (m, 1H),2.18-2.08 (m, 2H), 1.96 (dd, J=2.4, 13.7 Hz, 1H), 1.88 (dd, J=5.1, 17.8Hz, 1H), 1.82-1.70 (m, 2H), 1.67-1.57 (m, 3H), 1.49 (d, J=17.6 Hz, 1H),1.20-1.08 (m, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₂NaO₄NCl [M+Na]⁺:528.1918. found 528.1929.

Synthesis of Isoquinoline Compound 12

A solution of 1-chloroisoquinoline adduct 11 (115 mg, 227 μmol, 1.00equiv) in dichloromethane (20 mL) was cooled to 0° C. Pyridine (183 μL,2.30 mmol, 10.0 equiv) and DMAP (13.9 mg, 114 μmol, 0.50 equiv) werethen added sequentially to the solution. After 5 min, trifluoroaceticanhydride (158 μL, 1.14 mmol, 5.00 equiv) was added dropwise and stirredadditional 30 min, at which point pH 7 phosphate buffer (15 mL) wasadded followed by warming the reaction flask to room temperature. Theorganic and aqueous layers were separated and the aqueous layer wasextracted with dichloromethane (2×15 mL). The organic layers werecombined, washed with brine (25 mL), dried over Na₂SO₄, and concentratedunder reduced pressure. The resulting residue was then purified by shortflash column chromatography (silica gel, eluent: 2:1 Hexanes:EtOAc) toafford trifluoroacetylated product which was quickly used for the nextstep.

Trifluoroacetylated product (130 mg, 216 mmol, 1.00 equiv) wasazeotropically dried with benzene and dissolved in benzene (4.3 mL).AIBN (106 mg, 647 μmol, 3.00 equiv) was added and the reaction flask wasdegassed by the freeze-pump thaw process (3 cycles). Bu₃SnH (1.16 mL,4.31 mmol, 20.0 equiv) was added and the reaction mixture was allowed towarm to reflux. After 3 h, the reaction mixture was cooled to roomtemperature and concentrated under reduced pressure. The resultingresidue was then purified by flash column chromatography (silica gel,eluent: 4:1→3:1→1:1 Hexanes:EtOAc) to provide isoquinoline 12 (67.0 mg,65% in two steps). See also Yamashita et al., J. Org. Chem. 2011, 76,2408-2425.

¹H NMR (500 MHz, CDCl₃) Shift=9.21 (s, 1H), 8.46 (d, J=5.9 Hz, 1H), 7.77(s, 1H), 7.73 (d, J=8.3 Hz, 1H), 7.61 (d, J=5.9 Hz, 1H), 7.57 (d, J=8.3Hz, 1H), 5.74 (s, 1H), 5.29-5.23 (m, 1H), 4.00-3.90 (m, 4H), 3.11 (t,J=10.0 Hz, 1H), 2.49 (dd, J=8.3, 11.2 Hz, 1H), 2.47-2.41 (m, 1H),2.38-2.24 (m, 4H), 2.24-2.14 (m, 2H), 2.12 (d, J=13.2 Hz, 1H), 2.06-1.95(m, 2H), 1.91 (dd, J=5.4, 17.6 Hz, 1H), 1.83 (dq, J=4.9, 11.7 Hz, 1H),1.77 (td, J=2.3, 12.9 Hz, 1H), 1.72-1.59 (m, 3H), 0.52 (s, 3H). HRMS(ESI) (m/z) calc'd for C₃₀H₃₃NaNO₃ [M+Na]⁺: 478.2353. found 478.2347.

Synthesis of Ketone 13

To a solution of isoquinoline 12 (19.0 mg, 41.7 μmol, 1.00 equiv) inacetone (1.4 mL) and water (350 μL) was added PTSA (20.9 mg, 83.4 μmol,2.00 equiv) and the reaction mixture was warmed to 55° C. After 14.5 h,the reaction was cooled to room temperature and saturated NaHCO₃solution (2 mL) and ethyl acetate (2.5 mL) were sequentially added tothe reaction. The layers were separated and the aqueous layer wasextracted with ethyl acetate (2×2.5 mL). The organic layers werecombined, washed with brine (2 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The resulting residue was then purified by flashchromatography (silica gel, eluent: 3:2→1:2 Hexanes:EtOAc) to affordketone 13 (15.0 mg, 87%).

¹H NMR (500 MHz, CDCl₃) Shift=9.23 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.80(s, 1H), 7.78 (d, J=8.3 Hz, 1H), 7.65 (d, J=5.9 Hz, 1H), 7.61 (d, J=8.3Hz, 1H), 5.91 (s, 1H), 5.40-5.35 (m, 1H), 3.15 (t, J=10.0 Hz, 1H), 2.94(d, J=15.1 Hz, 1H), 2.68 (d, J=15.1 Hz, 1H), 2.67-2.59 (m, 1H),2.58-2.41 (m, 4H), 2.41-2.24 (m, 3H), 2.24-2.10 (m, 2H), 2.04 (tt,J=4.6, 13.2 Hz, 1H), 1.96 (dd, J=5.4, 17.6 Hz, 1H), 1.86 (dq, J=5.1,12.1 Hz, 1H), 1.80-1.67 (m, 2H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'dfor C₂₈H₃₀NO₂ [M+H]⁺: 412.2271, found 412.2288.

Epoxy Alcohol Compounds 3 and 3a

To a solution of estrone (180 g, 667 mmol, 1.00 equiv) in DMSO (2.8 L)was added KOH pellet (85% technical grade, 150 g, 2.26 mol, 3.40 equiv)and CH₃I (83.0 mL, 1.33 mol, 2.00 equiv). The reaction mixture wasstirred for 3.5 hours at room temperature and distilled water (2 L) wasslowly added at 0° C. The aqueous layer was extracted withdichloromethane (3×1.5 L) and the combined organic layer was washed withbrine (1.5 L). The organic layer was concentrated under nitrogen flow togive white crystalline, which was washed with cold methanol. The 180 gof crude mixture was used in the next step without further purification.The rest of three steps were conducted in two batches.

To a solution of the crude mixture (100 g, 352 mmol, 1.00 equiv) inmethanol (750 mL) and dichloromethane (750 mL) was added NaHCO₃ (93.8 g,1.05 mmol, 3.00 equiv). DDQ (120 g, 527 mmol, 1.50 equiv) was added infour portions with 5 min interval and the reaction mixture was stirredfor 2 hours and then quenched with the 10% aqueous Na₂S₂O₃ (500 mL). Thereaction flask was stirred for additional 30 min and filtered throughcelite, washed with chloroform. The 2 M NaOH solution (500 mL) was addedand the organic and aqueous layers were separated and the aqueous phasewas extracted with chloroform (3×700 mL). The combined organic phaseswere washed with brine (700 mL) and dried (Na₂SO₄). The solvent wasevaporated under reduced pressure and the 89 g of crude mixture was usedin the next step without further purification.

To a solution of the crude mixture (81.4 g, 291 mmol, 1.00 equiv) inbenzene (1.25 L) was added ethylene glycol (162 mL, 2.91 mol, 10 equiv)and PTSA (11.1 g, 58.3 mmol, 0.20 equiv). The reaction mixture waswarmed to reflux and water was trapped by Dean-Stark apparatus. After 24hours, the reaction was allowed to cool to room temperature andsaturated NaHCO₃ solution (500 mL) was applied. The aqueous phase wasextracted with ethyl acetate (3×300 mL) and the combined organic phaseswere washed with brine (500 mL). The organic phase was dried (Na₂SO₄)and the solvent was evaporated under reduced pressure. The 81.4 g ofcrude product was used in the next step without further purification.

To a solution of mixture of 8,9 and 9,11-unsaturated ethyleneketals(81.4 g, 249 mmol, 1.00 equiv) in dichloromethane (1.28 L) was addedmagnesium monoperoxyphthalate hexahydrate (354 g, 573 mmol, 2.30 equiv)and water (25 mL). The reaction mixture was stirred for 16 hours at roomtemperature and then filtered through celite pad. To the filtrate wasadded saturated NaHCO₃ solution (800 mL) and the organic and aqueouslayers were separated and the aqueous phase was extracted withdichloromethane (3×700 mL). The combined organic phases were washed withbrine (700 mL) and dried (Na₂SO₄). The solvent was evaporated underreduced pressure and the residue was purified by flash chromatography(silica gel, eluent: 3:1→2:1 Hexanes:EtOAc) to provide epoxy alcohol 3and 3a (3:3a=8:1, 81.0 g from 180 g, 34% in 4 steps). ¹H NMR and ¹³C NMRwere analyzed for epoxy alcohol 3.

¹H NMR (500 MHz, CDCl₃) Shift=7.77 (d, J=8.3 Hz, 1H), 6.76 (dd, J=2.0,8.3 Hz, 1H), 6.63 (d, J=2.0 Hz, 1H), 4.78 (dd, J=7.8, 9.8 Hz, 1H),3.95-3.87 (m, 4H), 3.78 (s, 3H), 2.84 (dt, J=5.9, 14.4 Hz, 1H), 2.49(dd, J=4.4, 15.1 Hz, 1H), 2.36-2.29 (m, 1H), 2.26 (dd, J=5.9, 14.2 Hz,2H), 2.06 (t, J=11.7 Hz, 1H), 1.97 (dd, J=7.3, 12.2 Hz, 1H), 1.94-1.88(m, 2H), 1.75 (dt, J=5.4, 14.2 Hz, 1H), 1.63-1.53 (m, 1H), 1.46 (t,J=11.0 Hz, 1H), 0.75 (s, 3H); ¹³C NMR (500 MHz, CDCl₃) Shift=159.1,139.5, 130.2, 125.9, 117.6, 114.3, 111.1, 69.0, 67.5, 65.4, 64.5, 61.8,55.2, 47.4, 46.8, 37.0, 34.9, 26.2, 25.0, 21.3, 14.9; FTIR (cm⁻¹) 3464,2944, 2885, 1610, 1503; HRMS (ESI) (m/z) calc'd for C₂₁H₂₇O₅ [M+H]⁺:359.1853. found 359.1852; [α]_(D)=−147.4° (c=0.01 g/mL in CHCl₃).

Diol Compound 5

To a solution of 3 and 3a (10.0 g, 27.9 mmol, 1.00 equiv) in1,2-dichloroethane (175 mL) was added NaBH₃CN (3.51 g, 55.8 mmol, 2.00equiv) and AcOH (3.19 mL, 55.8 mmol, 2.00 equiv) sequentially at roomtemperature. After 2.5 hours, saturated NaHCO₃ solution (150 mL) wasadded and the organic and aqueous layers were separated. The aqueousphase was extracted with dichloromethane (3×150 mL). The combinedorganic phases were washed with brine (200 mL), dried over Na₂SO₄, andconcentrated under reduced pressure. The crude mixture was used for thenext step without further purification.

Ammonia gas was condensed (60 mL) and to the liquid ammonia was added Li(1.94 g, 279 mmol, 10.0 equiv) at −78° C. After stirring for 30 min, thecrude mixture in THF (10 mL) and EtOH (6 mL) was cannulated and thereaction was warmed up to −40° C. After 1.5 hours, to the reactionmixture was added EtOH (20 mL) and EtOH:H₂O=1:1 (20 mL) sequentially,and the flask was opened to gently evaporate liquid ammonia by removingthe cooling bath. Water (50 mL) was added and the aqueous phase wasextracted with diethyl ether (3×90 mL). The combined organic phases werewashed with brine (150 mL), dried (Na₂SO₄), and concentrated underreduced pressure. The product was used in the next step without furtherpurification.

To a solution of Birch reduction product in THF (300 mL) and ethyleneglycol (75 mL) was added PTSA (530 mg, 2.79 mmol, 0.100 equiv). Thereaction mixture was stirred for 30 min at room temperature andsaturated NaHCO₃ solution (200 mL) was added. The organic and aqueouslayers were separated and the aqueous phase was extracted withchloroform (4×200 mL). The combined organic phases were washed withbrine (200 mL) and dried (Na₂SO₄). The solvent was evaporated underreduced pressure and the residue was purified by flash chromatography(silica gel, eluent: 4:1 Hexanes:EtOAc→100% EtOAc→10:1 EtOAc:MeOH) toprovide diol 5 (6.6 g, 60% in 3 steps).

¹H NMR (500 MHz, C₆D₆) Shift=3.67-3.42 (m, 9H), 3.25-3.14 (m, 1H), 2.40(dd, J=5.9, 13.2 Hz, 1H), 2.31 (br. s, 2H), 2.23-2.09 (m, 2H), 2.03 (t,J=10.7 Hz, 1H), 1.97-1.90 (m, 2H), 1.89 (dd, J=8.3, 14.2 Hz, 1H),1.85-1.75 (m, 4H), 1.66-1.50 (m, 4H), 1.00 (s, 3H); ¹³C NMR (500 MHz,CDCl₃) Shift=127.8, 126.9, 118.3, 108.5, 74.2, 72.0, 65.3, 64.7, 64.4,64.3, 63.7, 58.9, 52.5, 45.9, 41.2, 40.5, 33.8, 31.6, 27.6, 25.8, 18.0,16.6; FTIR (cm⁻¹) 3417, 2940, 2884; HRMS (ESI) (m/z) calc'd forC₂₂H₃₂NaO₆ [M+Na]⁺: 415.2091. found 415.2076; [α]_(D)=−13.4° (c=0.01g/mL in CHCl₃).

Allylic Alcohol Compound 7

To a solution of diol 5 (4.05 g, 10.3 mmol, 1.00 equiv) indichloromethane (230 mL) was added NBS (2.00 g, 11.4 mmol, 1.10 equiv)at one portion at −10 OC and the reaction mixture was warmed to roomtemperature. The reaction was monitored by TLC (about 30 min for thecompletion). Once the reaction is done, the reaction mixture was cooledto −40° C. and triethylamine (17.3 mL, 124 mmol, 12.0 equiv) was added.Pre-stirred SO₃ Py (16.4 g, 103 mmol, 10.0 equiv) in DMSO (115 mL) for20 min at room temperature was added to the reaction mixture at −40° C.,which was subsequently allowed to warm slowly to room temperature. After3 hours, saturated NH₄Cl solution (130 mL) was added and the reactionwas allowed to warm to room temperature. The organic and aqueous layerswere separated and the aqueous phase was extracted with dichloromethane(2×200 mL). The combined organic phases were washed with brine (150 mL),dried over Na₂SO₄, and concentrated under reduced pressure. The crudemixture was used without further purification.

The crude mixture was dissolved in dichloromethane (300 mL) and thereaction mixture was cooled to −40° C. followed by the slow addition ofDBU (3.90 mL, 25.6 mmol, 2.50 equiv). After 15 min, saturated NH₄Clsolution (130 mL) was added and the reaction was allowed to warm to roomtemperature. The organic and aqueous layers were separated and theaqueous phase was extracted with dichloromethane (2×200 mL). Thecombined organic phases were washed with brine (150 mL), dried overNa₂SO₄, and concentrated under reduced pressure.

To a solution of crude mixture (3.20 g, 8.32 mmol, 1.00 equiv) in MeOH(150 mL) and THF (20 mL) was added CeCl₃.7H₂O (9.20 g, 24.7 mmol, 3.00equiv) at room temperature. After stirring 5 min, the reaction wascooled to −20° C. followed by the addition of NaBH₄ (623 mg, 16.5 mmol,2.00 equiv). After 30 min, saturated NH₄Cl solution (50 mL) and water(50 mL) was added, which was allowed to warm to room temperature. Theaqueous phase was extracted with ethyl acetate (3×200 mL) and thecombined organic phases were washed with brine (150 mL), dried overNa₂SO₄, and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, eluent: 20:1 DCM:MeOH) toafford allylic alcohol 7 (2.60 g, 64% in 4 steps).

¹H NMR (500 MHz, C₆D₆) Shift=4.39-4.30 (m, 1H), 3.58-3.36 (m, 8H), 3.22(dd, J=3.7, 16.4 Hz, 1H), 2.94 (dd, J=7.1, 12.5 Hz, 1H), 2.66 (d, J=13.2Hz, 1H), 2.49-2.41 (m, 1H), 2.39 (dd, J=2.2, 12.9 Hz, 1H), 2.07-1.99 (m,1H), 1.96-1.79 (m, 6H), 1.73 (br. s, 3H), 1.66-1.57 (m, 1H), 1.15-1.07(m, 1H), 0.86 (s, 3H); ¹³C NMR (500 MHz, C₆D₆) Shift=140.6, 139.1,118.7, 109.5, 88.3, 86.2, 67.1, 65.4, 64.6, 64.2, 47.9, 46.5, 41.3,40.9, 34.7, 34.2, 33.9, 30.0, 20.4, 19.8, 15.6; HRMS (ESI) (m/z) calc'dfor C₂₂H₃₀NaO₆ [M+Na]⁺: 413.1935, found 413.1942.

Reductive Amination Method A

To a solution of ketone 13 (1.00 equiv) in 1,2-dichloroethane (0.02 M)was sequentially added amine (4.00 equiv), AcOH (1.50 equiv), andNaBH₃CN (3.50 equiv) at room temperature. Triethylamine (4 equiv) wasadded if the reacting amine is a form of HCl salt (Method AA). Once thereaction is done, saturated NaHCO₃ solution was added and the layerswere separated. The aqueous layer was extracted with dichloromethane.The organic layers were combined, washed with brine, dried over Na₂SO₄and concentrated under reduced pressure. (α-NR₂:β-NR₂=˜1:1.2 to ˜1:5).

Method B

To a solution of ketone 13 (1.00 equiv) in 1,2-dichloroethane (0.02 M)was sequentially added amine (2.00 equiv), AcOH (2.00 equiv), andNaBH(OAc)₃ (2.00 equiv) at room temperature. Triethylamine (2.00 equiv)was added if the reacting amine is a form of HCl salt (Method BB). Oncethe reaction is done, saturated NaHCO₃ solution was added and the layerswere separated. The aqueous layer was extracted with dichloromethane.The organic layers were combined, washed with brine, dried over Na₂SO₄and concentrated under reduced pressure. (α-NR₂:β-NR₂=˜1:1.2 to ˜1:5).

Method C

To a solution of secondary amine (1.00 equiv) in dichloromethane (0.02M) was added formaldehyde or acetaldehyde (5.00 equiv) and stirred 1 hat room temperature before the addition of NaBH(OAc)₃ (2.00 equiv). Oncethe reaction is done, saturated NaHCO₃ solution was added and the layerswere separated. The aqueous layer was extracted with dichloromethane.The organic layers were combined, washed with brine, dried over Na₂SO₄and concentrated under reduced pressure.

Method D: General Method for Favoring α-Amine

To a solution of ketone 13 (1.00 equiv) in THF and t-BuOH (4:1, 0.02 M)was added amine (5.00 equiv) and Ti(Oi-Pr)₄ (3.00 equiv) sequentially,and stirred at room temperature for 15 hours (4 hours for Me₂NH, MeNH₂,and NH₃). The reaction mixture was cooled to −20° C. and NaBH₄ (1.50equiv) was added. Once the reaction is done, saturated NaHCO₃ solutionwas added and the layers were separated. The aqueous layer was extractedwith EtOAc. The organic layers were combined, washed with brine, driedover Na₂SO₄ and concentrated under reduced pressure. (α-NR₂:β-NR₂=˜1.1:1to ˜3.7:1).

Method E: General Method for Methanesulfonamide Formation

To a solution of amine (1.00 equiv) in dichloromethane (0.013 M) wasadded trimethylamine (4.00 equiv) and the reaction mixture was cooled to−20° C. Methanesulfonic anhydride (2.50 equiv) was added as a solutionin dichloromethane and stirred 30 min at the same temperature. 2 N NaOHsolution was added and the layers were separated. The aqueous layer wasextracted with dichloromethane. The organic layers were combined, washedwith brine, dried over Na₂SO₄ and concentrated under reduced pressure.

β-Dimethylamine 14B and α-Dimethylamine 14A

The crude mixture was purified sequentially by flash chromatography(silica gel, eluent: 20:1 EtOAc:2M NH₃ solution in MeOH) to affordβ-dimethylamine 14B (21.5 mg, 65%). ca. 0.6 mg of α-dimethylamine 14Awas prepared from 3 mg of 13 by HPLC (Eclipse XDB-C8 column, 9.4 mm×25cm; gradient=0%→35% MeCN (0.1% formic acid):H₂O (0.1% formic acid) over30 min)

β-dimethylamine 14B:

¹H NMR (500 MHz, C₆D₆) Shift=9.31 (s, 1H), 8.61 (d, J=5.4 Hz, 1H), 7.43(s, 1H), 7.39 (d, J=8.8 Hz, 1H), 7.25 (d, J=5.4 Hz, 1H), 7.23 (d, J=8.8Hz, 1H), 5.73 (br. s, 1H), 5.18 (s, 1H), 2.74 (t, J=10.0 Hz, 1H), 2.63(dd, J=8.8, 11.2 Hz, 1H), 2.48-2.28 (m, 2H), 2.27-2.20 (m, 1H),2.19-2.03 (m, 6H), 2.00 (br. s, 6H), 1.95-1.84 (m, 2H), 1.83-1.66 (m,5H), 1.41 (tt, J=5.4, 13.2 Hz, 1H), 0.45 (s, 3H). HRMS (ESI) (m/z)calc'd for C₃₀H₃₇N₂O [M+H]⁺: 441.2900. found 441.2910.

α-dimethylamine 14A:

¹H NMR (600 MHz, C₆D₆) Shift=9.26 (s, 1H), 8.56 (d, J=5.9 Hz, 1H),7.44-7.39 (m, 1H), 7.36 (d, J=8.2 Hz, 1H), 7.21-7.20 (m, 1H), 7.20 (d,J=5.9 Hz, 1H), 5.68-5.65 (m, 1H), 5.15-5.11 (m, 1H), 2.72-2.66 (m,J=10.0 Hz, 1H), 2.59 (dd, J=8.8, 11.2 Hz, 1H), 2.34 (tt, J=2.9, 12.1 Hz,1H), 2.16 (td, J=3.2, 16.0 Hz, 1H), 2.09 (s, 6H), 2.13-1.92 (m, 8H),1.85 (ddd, J=5.0, 9.0, 13.6 Hz, 1H), 1.73 (dt, J=5.3, 12.3 Hz, 1H),1.72-1.66 (m, 2H), 1.60-1.57 (m, 1H), 1.57-1.49 (m, 1H), 1.20 (dq,J=4.1, 12.3 Hz, 1H), 0.40 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₇N₂O[M+H]⁺: 441.2900. found 441.2909.

β-Morpholine 15B and α-Morpholine 15A

β-Morpholine 15B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 100% EtOAc→35:1→20:1→10:1 EtOAc:MeOH) to afford β-morpholine 15B(21 mg, 66%). ¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.9Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.3 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H),7.59 (dd, J=1.0, 8.8 Hz, 1H), 5.71 (s, 1H), 5.24 (d, J=2.9 Hz, 1H), 3.73(br. s, 4H), 3.13 (t, J=10.0 Hz, 1H), 2.65-2.28 (m, 11H), 2.23-2.11 (m,3H), 2.06 (d, J=13.2 Hz, 1H), 2.01 (dt, J=4.4, 9.0 Hz, 1H), 1.93 (dd,J=4.9, 17.1 Hz, 1H), 1.89-1.79 (m, 1H), 1.75-1.53 (m, 4H), 0.54 (s, 3H).HRMS (ESI) (m/z) calc'd for C₃₂H₃₉N₂O₂ [M+H]⁺: 483.3006. found 483.3012.

β-N-Methylpiperazine 16B and α-N-Methylpiperazine 16A

β-N-Methylpiperazine 16B:

The crude mixture was purified sequentially by flash chromatography(silica gel, 1^(st) column: eluent: 100% MeOH→10:1 EtOAc:2M NH₃ solutionin MeOH/2^(nd) column: eluent: 20:1 EtOAc:2M NH₃ solution in MeOH)) toafford β-N-methylpiperazine 16B (20 mg, 55%). ¹H NMR (600 MHz, CDCl₃)Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.8Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.59 (d, J=8.2 Hz, 1H), 5.70 (s, 1H),5.25-5.22 (m, 1H), 3.13 (t, J=9.7 Hz, 1H), 2.53 (br. s., 1H), 2.50 (dd,J=8.8, 11.7 Hz, 1H), 2.41 (t, J=12.9 Hz, 1H), 2.38-2.33 (m, 3H), 2.32(br. s, 3H), 2.22-2.11 (m, 3H), 2.10-1.95 (m, 3H), 1.95-1.89 (m, 2H),1.84 (dq, J=5.3, 11.7 Hz, 1H), 1.79-1.50 (m, 11H), 0.62-0.43 (m, 3H).HRMS (ESI) (m/z) calc'd for C₃₃H₄₂N₃O [M+H]⁺: 496.3322. found 496.3337.

β-Azetidine 18B and α-Azetidine 18A

β-Azetidine 18B:

The crude mixture was purified by preparative TLC (eluent: 1:1EtOAc:MeOH) to afford β-azetidine 18B (2.7 mg, 50%). ¹H NMR (500 MHz,CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79 (s, 1H), 7.75(d, J=8.8 Hz, 1H), 7.62 (d, J=5.4 Hz, 1H), 7.59 (d, J=8.3 Hz, 1H), 5.69(s, 1H), 5.22 (d, J=2.4 Hz, 1H), 3.20-3.05 (m, 5H), 2.59-2.43 (m, 4H),2.39-2.28 (m, 2H), 2.23-2.12 (m, 2H), 2.07-1.96 (m, 4H), 1.92 (dd,J=5.1, 17.3 Hz, 1H), 1.89-1.79 (m, 3H), 1.75-1.55 (m, 3H), 1.40 (t,J=13.2 Hz, 1H), 0.54 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₁H₃₇N₂O[M+H]⁺: 453.2906. found 453.2916.

β-Pyrrolidine 19B and α-Pyrrolidine 19A

β-Pyrrolidine 19B:

The crude mixture was purified by preparative TLC (eluent: 20:10:3EtOAc:Hexane: 2M NH₃ solution in MeOH) to afford β-pyrrolidine 19B (2.0mg, 40%). ¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.4 Hz,1H), 7.79 (s, 1H), 7.75 (d, J=8.3 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.59(d, J=8.3 Hz, 1H), 5.70 (br. s., 1H), 5.22 (br. s., 1H), 3.13 (t, J=9.8Hz, 1H), 2.59-2.46 (m, 6H), 2.44 (br. s., 1H), 2.41-2.28 (m, 3H),2.23-2.12 (m, 2H), 2.11-2.00 (m, 2H), 2.00-1.82 (m, 4H), 1.79-1.65 (m,6H), 1.64-1.51 (m, 2H), 0.54 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₂H₃₉N₂O [M+H]⁺: 467.3057. found 467.3053.

β-Dimethylamine 17,18-Unsaturated Isoquinoline 23B and α-Dimethylamine17,18-Unsaturated Isoquinoline 23A

β-Dimethylamine 17,18-Unsaturated Isoquinoline 23B:

The crude mixture was purified sequentially by flash chromatography(silica gel, eluent: 20:1 EtOAc:2M NH₃ solution in MeOH) to affordβ-dimethylamine 17,18-unsaturated isoquinoline 23B (6.5 mg, 74%). ¹H NMR(500 MHz, CDCl₃) Shift=9.24 (br. s., 1H), 8.51 (d, J=5.4 Hz, 1H), 7.94(s, 1H), 7.84-7.76 (m, 2H), 7.63 (d, J=5.4 Hz, 1H), 6.27 (br. s., 1H),5.97 (s, 1H), 5.50 (dd, J=2.4, 4.9 Hz, 1H), 2.98 (d, J=14.6 Hz, 1H),2.78 (dd, J=6.8, 11.2 Hz, 1H), 2.71 (d, J=14.6 Hz, 1H), 2.72-2.63 (m,1H), 2.61 (d, J=5.4 Hz, 1H), 2.59-2.54 (m, 2H), 2.54-2.50 (m, 2H),2.50-2.42 (m, 2H), 2.39 (ddd, J=1.5, 11.0, 12.9 Hz, 1H), 2.20 (ddd,J=1.5, 9.5, 11.5 Hz, 1H), 2.01 (ddd, J=7.3, 8.8, 12.7 Hz, 1H), 1.79 (dt,J=7.3, 11.2 Hz, 1H), 1.18 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₅N₂O[M+H]⁺: 439.2744. found 439.2753.

β-Monomethylamine 24B and α-Monomethylamine 24A

β-Monomethylamine 24B:

The crude mixture was purified by preparative TLC (eluent: 10:1 EtOAc:2MNH₃ solution in MeOH) to afford β-monomethylamine 24B (ca. 1.5 mg, 58%).¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.4 Hz, 1H), 7.79(s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.59 (dd,J=1.0, 8.3 Hz, 1H), 5.72 (d, J=1.0 Hz, 1H), 5.24 (dd, J=2.2, 5.1 Hz,1H), 3.13 (t, J=10.0 Hz, 1H), 3.03-2.98 (m, 1H), 2.57-2.50 (m, 1H), 2.51(dd, J=8.3, 11.7 Hz, 1H), 2.44 (s, 3H), 2.36 (d, J=15.2 Hz, 1H),2.36-2.28 (m, 2H), 2.26-2.13 (m, 2H), 2.09 (dd, J=3.7, 16.4 Hz, 1H),2.07-1.99 (m, 2H), 1.98-1.92 (m, 1H), 1.93 (dd, J=5.9, 17.6 Hz, 1H),1.85 (dq, J=4.9, 11.7 Hz, 1H), 1.82-1.76 (m, 1H), 1.76-1.58 (m, 3H),0.54 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₉H₃₅N₂O [M+H]⁺: 427.2744.found 427.2740.

β-Deuterodimethylamine 26B and α-Deuterodimethylamine 26A

β-Deuterodimethylamine 26B:

Triethylamine was added The crude mixture was purified sequentially byflash chromatography (silica gel, eluent: 20:1 EtOAc:2M NH₃ solution inMeOH) to afford β-deuterodimethylamine 26B (4 mg, 62%). ¹H NMR (500 MHz,CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz, 1H), 7.80 (s, 1H), 7.77(d, J=8.3 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 5.74(br. s., 1H), 5.26 (br. s., 1H), 3.15 (t, J=10.0 Hz, 1H), 2.51 (dd,J=8.8, 11.2 Hz, 1H), 2.50-2.42 (m, 1H), 2.37 (d, J=17.1 Hz, 1H),2.38-2.26 (m, 2H), 2.26-2.09 (m, 4H), 2.08-1.98 (m, 2H), 1.95 (dd,J=5.1, 17.3 Hz, 1H), 1.87 (dq, J=5.4, 12.2 Hz, 1H), 1.80-1.68 (m, 3H),1.62 (br. s., 2H), 0.63-0.50 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₀H₃₁D₆N₂O [M+H]⁺: 447.3277. found 447.3281.

β-2-Methoxyethylmethylamine 27B and α-2-Methoxyethylmethylamine 27A

β-2-Methoxyethylmethylamine 27B:

The crude mixture was purified by preparative TLC (eluent: 10:10:1Hexanes:EtOAc:2M NH₃ solution in MeOH) to affordβ-2-methoxyethylmethylamine 27B (ca. 1.2 mg, 20%). ¹H NMR (500 MHz,CD₃OD) Shift=9.21 (s, 1H), 8.40 (d, J=5.4 Hz, 1H), 7.99 (s, 1H), 7.90(d, J=8.8 Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.77 (s, 1H), 5.80 (s, 1H),5.35-5.27 (m, 1H), 3.60 (t, J=5.4 Hz, 2H), 3.39 (s, 2H), 3.24 (t, J=10.0Hz, 1H), 3.15-2.88 (m, 2H), 2.56 (br. s., 3H), 2.51 (dd, J=9.3, 10.7 Hz,1H), 2.48-2.39 (m, 3H), 2.35-2.28 (m, 1H), 2.25-2.09 (m, 4H), 2.02-1.84(m, 7H), 1.76 (s, 2H), 0.59 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₂H₄₁N₂O₂ [M+H]⁺: 485.3163. found 485.3170.

β-Bis-2-Methoxyethylamine 28B and α-Bis-2-Methoxyethylamine 28A

β-Bis-2-Methoxyethylamine 28B:

The crude mixture was purified by preparative TLC (eluent: 10:1Dichloromethane:MeOH) to afford β-bis-2-methoxyethylamine 28B (ca. 1.1mg, 19%). ¹H NMR (500 MHz, CD₃OD) Shift=9.21 (s, 1H), 8.39 (d, J=5.9 Hz,1H), 7.99 (s, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.77(s, 1H), 5.74 (s, 1H), 5.29-5.24 (m, 1H), 3.47 (t, J=6.1 Hz, 4H), 3.36(s, 6H), 3.24 (t, J=10.5 Hz, 1H), 3.06-2.93 (m, 1H), 2.78 (d, J=5.9 Hz,4H), 2.52 (dd, J=9.0, 11.5 Hz, 1H), 2.49-2.43 (m, 1H), 2.44 (d, J=17.6Hz, 1H), 2.40-2.35 (m, 1H), 2.35-2.26 (m, 1H), 2.24-2.10 (m, 3H),2.07-1.94 (m, 3H), 1.91 (dd, J=5.4, 17.6 Hz, 1H), 1.85 (d, J=14.6 Hz,1H), 1.82-1.67 (m, 3H), 0.59 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₄H₄₅N₂O₃ [M+H]⁺: 529.3425. found 529.3434.

β-2-Fluoroethylmethylamine 29B and α-2-Fluoroethylmethylamine 29A

β-2-Fluoroethylmethylamine 29B:

The crude mixture was purified by preparative TLC (eluent: 20:1Dichloromethane:MeOH) to afford β-2-fluoroethylmethylamine 29B (2.7 mg,51%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz,1H), 7.80 (s, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60(dd, J=1.0, 8.3 Hz, 1H), 5.74 (br. s., 1H), 5.26 (br. s., 1H), 4.68-4.46(m, 2H), 3.15 (t, J=9.8 Hz, 1H), 2.99-2.69 (m, 3H), 2.52 (dd, J=8.8,11.2 Hz, 1H), 2.47-2.30 (m, 6H), 2.29-2.16 (m, 4H), 2.16-2.00 (m, 3H),2.01-1.92 (m, 1H), 1.94 (dd, J=5.1, 17.3 Hz, 1H), 1.86 (dq, J=5.4, 12.2Hz, 1H), 1.79-1.64 (m, 3H), 0.56 (s, 3H). HRMS (ESI) (m z) calc'd forC₃₁H₃₈N₂OF [M+H]⁺: 473.2963, found 473.2971.

β-2,2-Difluoroethylmethylamine 30B and α-2,2-Difluoroethylmethylamine30A

β-2,2-Difluoroethylmethylamine 30B:

The crude mixture was purified by preparative TLC (eluent: 1:1Hexanes:EtOAc) to afford β-2,2-difluoroethylmethylamine 30B (ca. 1.1 mg,19%). ¹H NMR (500 MHz, CDCl₃) Shift=9.28 (s, 1H), 8.50 (d, J=5.9 Hz,1H), 7.86 (s, 1H), 7.83 (d, J=8.3 Hz, 1H), 7.75 (d, J=5.4 Hz, 1H), 7.69(d, J=8.3 Hz, 1H), 6.15-5.83 (m, 1H), 5.75 (s, 1H), 5.27 (dd, J=2.4, 5.4Hz, 1H), 3.17 (t, J=10.0 Hz, 1H), 2.91 (br. s., 2H), 2.52 (dd, J=8.8,11.7 Hz, 1H), 2.47 (br. s, 3H), 2.45-2.30 (m, 4H), 2.27-2.11 (m, 6H),2.11-1.99 (m, 2H), 1.95 (dd, J=5.1, 17.3 Hz, 1H), 1.88 (dq, J=6.3, 12.7Hz, 1H), 1.77-1.68 (m, 3H), 0.56 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₁H₃₇N₂OF₂ [M+H]⁺: 491.2868. found 491.2879.

β-7-Azabicyclo[2.2.1]Heptane 31B and α-7-Azabicyclo[2.2.1]Heptane 31A

β-7-Azabicyclo[2.2.1]heptane 31B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 10:10:1→10:10:2 Hexanes:EtOAc:2M NH₃ solution in MeOH) to affordβ-7-azabicyclo[2.2.1]heptane 31B (3 mg, 50%). ¹H NMR (500 MHz, CDCl₃)Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz, 1H), 7.81 (s, 1H), 7.77 (d, J=8.3Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.61 (d, J=8.3 Hz, 1H), 5.71 (br. s.,1H), 5.24 (br. s., 1H), 3.43 (br. s., 2H), 3.15 (t, J=9.8 Hz, 1H), 2.69(br. s., 2H), 2.52 (t, J=9.8 Hz, 1H), 2.46 (br. s., 1H), 2.37 (d, J=17.6Hz, 1H), 2.38-2.29 (m, 1H), 2.27-2.13 (m, 3H), 2.11-1.92 (m, 6H), 1.87(dq, J=5.9, 12.7 Hz, 1H), 1.86-1.79 (m, 1H), 1.78-1.60 (m, 8H), 1.55 (t,J=13.2 Hz, 1H), 0.56 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₄H₄₁N₂O[M+H]⁺: 493.3213. found 493.3224.

β-Isopropylamine 32B and α-Isopropylamine 32A

β-Isopropylamine 32B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 10:1 EtOAc:2M NH₃ solution in MeOH) to afford β-isopropylamine32B (5 mg, 70%). ¹H NMR (600 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d,J=5.9 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.9 Hz,1H), 7.59 (d, J=8.2 Hz, 1H), 5.71 (s, 1H), 5.24 (d, J=2.9 Hz, 1H), 3.24(br. s., 1H), 3.13 (t, J=9.7 Hz, 1H), 2.90 (br. s., 1H), 2.50 (dd,J=8.5, 11.4 Hz, 2H), 2.41-2.26 (m, 3H), 2.24-2.13 (m, 3H), 2.09 (dd,J=2.9, 15.3 Hz, 1H), 2.06-1.98 (m, 2H), 1.93 (dd, J=5.3, 17.6 Hz, 1H),1.85 (dq, J=5.3, 12.3 Hz, 1H), 1.75-1.62 (m, 4H), 1.07 (br. s., 6H),0.54 (s, 3H). HRMS (ESI) (m z) calc'd for C₃₁H₃₉N₂O [M+H]⁺: 455.3057.found 493.3049.

β-Isopropylmethylamine 33B

The crude mixture was purified by preparative TLC (eluent: 20:10:3Hexanes:EtOAc:2M NH₃ solution in MeOH) to afford β-isopropylmethylamine33B (4 mg, 78%). ¹H NMR (600 MHz, CDCl₃) Shift=9.22 (br. s., 1H), 8.49(d, J=4.7 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.3Hz, 1H), 7.59 (d, J=8.2 Hz, 1H), 5.70 (br. s., 1H), 5.22 (br. s., 1H),3.22 (br. s., 1H), 3.13 (t, J=10.0 Hz, 1H), 2.77 (br. s., 1H), 2.50 (t,J=8.2 Hz, 1H), 2.43 (t, J=12.9 Hz, 1H), 2.36 (d, J=17.6 Hz, 1H),2.35-2.28 (m, 2H), 2.22-2.14 (m, 2H), 2.17 (dt, J=4.4, 9.0 Hz, 1H), 2.11(br. s., 3H), 2.08-1.99 (m, 2H), 1.98-1.91 (m, 1H), 1.93 (dd, J=4.1,17.0 Hz, 1H), 1.85 (dq, J=5.3, 12.3 Hz, 1H), 1.69 (br. s., 2H), 1.59(br. s., 1H), 1.56 (br. s., 1H), 0.96 (br. s., 6H), 0.54 (s, 3H). HRMS(ESI) (m/z) calc'd for C₃₂H₄₁N₂O [M+H]⁺: 455.3057. found 493.3049.

β-Isopropylethylamine 34B

The crude mixture was purified by preparative TLC (eluent: 100% MeOH) toafford β-isopropylethylamine 34B (ca. 1.5 mg, 20%). ¹H NMR (600 MHz,CD₃OD) Shift=9.19 (s, 1H), 8.38 (d, J=5.9 Hz, 1H), 7.97 (s, 1H), 7.88(d, J=8.8 Hz, 1H), 7.79 (d, J=5.9 Hz, 1H), 7.74 (d, J=8.2 Hz, 1H), 5.76(br. s., 1H), 5.27 (br. s., 1H), 3.26-3.19 (m, 1H), 2.91-2.66 (m, 3H),2.50 (dd, J=9.4, 11.2 Hz, 1H), 2.48-2.36 (m, 3H), 2.29 (t, J=10.6 Hz,1H), 2.23-2.06 (m, 4H), 2.00-1.86 (m, 5H), 1.86-1.79 (m, 1H), 1.79-1.66(m, 2H), 1.21-1.08 (m, 9H), 0.57 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₃H₄₃N₂O [M+H]⁺: 483.3370. found 483.3382.

β-(R)-3-Fluoropyrrolidine 35B and α-(R)-3-Fluoropyrrolidine 35A

β-(R)-3-Fluoropyrrolidine 35B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-(R)-3-fluoropyrrolidine 35B (2.2 mg, 38%). ¹H NMR (500 MHz, CDCl₃)Shift=δ 9.22 (s, 1H), 8.49 (d, J=5.4 Hz, 1H), 7.79 (s, 1H), 7.75 (d,J=8.8 Hz, 1H), 7.62 (d, J=5.4 Hz, 1H), 7.59 (d, J=8.8 Hz, 1H), 5.71 (d,J=2.0 Hz, 1H), 5.23 (m, 1H), 5.11 (m, 1H), 3.40 (m, 1H), 3.13 (dd,J=9.3, 9.3 Hz, 1H), 2.88-2.96 (m, 2H), 2.66-2.77 (m, 1H), 2.48-2.58 (m,3H), 2.29-2.45 (m, 3H), 2.12-2.23 (m, 3H), 1.99-2.09 (m, 5H), 1.83-1.97(m, 2H), 1.67-1.74 (m, 2H), 1.59 (m, 1H), 0.55 (s, 3H). HRMS (ESI) (m/z)calc'd for C₃₂H₃₈FN₂O [M+H]⁺: 485.2968. found 485.2915.

β-(S)-3-Fluoropyrrolidine 36B and α-(S)-3-Fluoropyrrolidine 36A

β-(S)-3-Fluoropyrrolidine 36B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-(S)-3-fluoropyrrolidine 36B (2.7 mg, 46%). ¹H NMR (500 MHz, CD₃OD)Shift=9.21-9.18 (m, 1H), 8.37 (d, J=5.87 Hz, 1H), 7.98-7.96 (m, 1H),7.88 (d, J=8.80 Hz, 1H), 7.80-7.77 (m, 1H), 7.74 (dd, J=8.56, 1.71 Hz,1H), 5.71 (d, J=1.47 Hz, 1H), 5.23 (m, 1H), 5.22-5.07 (m, 2H), 3.22 (t,J=9.8 Hz, 1H), 3.08-2.97 (m, 1H), 2.90 (td, J=8.19, 5.62 Hz, 1H),2.70-2.64 (m, 1H), 2.62-2.58 (m, 1H), 2.52-2.34 (m, 6H), 2.33-2.24 (m,1H), 2.23-2.03 (m, 3H), 2.03-1.85 (m, 6H), 1.77-1.67 (m, 2H), 1.67-1.56(m, 1H), 0.57 (s, 3H). HRMS (ESI) (m z) calc'd for C₃₂H₃₈FN₂O [M+H]⁺:485.6553. found 485.6551.

β-3,3-Difluoropyrrolidine 37B and α-3,3-Difluoropyrrolidine 37A

β-3,3-Difluoropyrrolidine 37B:

The crude mixture was purified by preparative TLC (eluent: 80:15:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-3,3-difluoropyrrolidine 37B (2.9 mg, 40%). ¹H NMR (500 MHz, CDCl₃)Shift=δ 9.22 (s, 1H), 8.49 (d, J=5.4 Hz, 1H), 7.79 (s, 1H), 7.75 (d,J=8.3 Hz, 1H), 7.62 (d, J=5.4 Hz, 1H), 7.59 (d, J=8.8 Hz, 1H), 5.72 (d,J=2.0 Hz, 1H), 5.25 (dd, J=5.4 Hz, 2.0 Hz, 1H), 3.13 (dd, J=9.3 Hz, 9.3Hz, 1H), 2.86-3.03 (m, 2H), 2.14 (dd, J=6.9, 6.9 Hz, 2H), 2.58 (m, 1H),2.50 (dd, J=11.7, 8.3 Hz, 2H), 2.13-2.44 (m, 6H), 1.98-2.10 (m, 2H),1.90-1.96 (m, 1H), 1.83-1.89 (m, 2H), 1.66-1.74 (m, 2H), 1.53-1.61 (m,1H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₂H₃₇F₂N₂O [M+H]⁺:503.2874 found 503.2814.

α-3,3-Difluoropyrrolidine 37A:

The crude mixture was purified by preparative TLC (eluent: 80:15:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordα-3,3-difluoropyrrolidine 37A (2.9 mg from 6.0 mg, 40%). ¹H NMR (500MHz, CDCl₃) Shift=δ 9.22 (s, 1H), 8.49 (d, J=5.4 Hz, 1H), 7.79 (s, 1H),7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.4 Hz, 1H), 7.59 (d, J=8.8 Hz, 1H),5.74 (s, 1H), 5.28 (d, J=2.4 Hz, 1H), 3.15 (dd, J=9.3 Hz, 9.3 Hz, 1H),3.01 (dt, J=13.7, 2.4 Hz, 2H), 2.83 (dd, J=6.8, 6.8 Hz, 2H), 2.52 (dd,J=11.2, 8.3 Hz, 2H), 2.15-2.40 (m, 6H), 2.02-2.07 (m, 3H), 1.79-1.97 (m,3H), 1.72 (m, 3H), 1.61 (m, 3H), 0.54 (s, 3H). HRMS (ESI) (m/z) calc'dfor C₃₂H₃₇F₂N₂O [M+H]⁺: 503.2874 found 503.2807.

β-2-Oxa-6-Azaspiro[3.4]Octane 38B and α-2-Oxa-6-Azaspiro[3.4]Octane 38A

β-2-oxa-6-azaspiro[3.4]octane 38B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-2-oxa-6-azaspiro[3.4]octane 38B (3.4 mg, 55%). ¹H NMR (500 MHz, CD₃OD)Shift=9.21 (s, 1H), 8.39 (d, J=5.4 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J=8.8Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.76 (dd, J=1.7, 8.5 Hz, 1H), 5.73-5.70(m, 1H), 5.28-5.23 (m, 1H), 4.63 (d, J=2.4 Hz, 4H), 3.28-3.21 (m, 1H),2.90 (dd, J=9.3, 49.8 Hz, 2H), 2.62 (t, J=7.3 Hz, 2H), 2.54-2.40 (m,5H), 2.36-2.27 (m, 2H), 2.23-2.16 (m, 1H), 2.13 (s, 2H), 2.10-2.05 (m,1H), 2.02-1.88 (m, 6H), 1.81-1.66 (m, 2H), 1.66-1.57 (m, 1H), 0.58 (s,3H). HRMS (ESI) (m z) calc'd for C₃₄H₄₁N₂O₂ [M+H]⁺: 509.7015. found509.7013.

β-Cyclopropylamine 39B and α-Cyclopropylamine 39A

β-Cyclopropylamine 39B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to afford β-cyclopropylamine 39B(3.7 mg, 55%). ¹H NMR (500 MHz, CD₃OD) Shift=9.21 (s, 1H), 8.39 (d,J=5.9 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.81 (d, J=5.4 Hz,1H), 7.76 (dd, J=1.7, 8.5 Hz, 1H), 5.73 (d, J=2.0 Hz, 1H), 5.28-5.24 (m,1H), 3.24 (t, J=20.0 Hz, 1H), 3.18-3.12 (m, 1H), 2.54-2.40 (m, 4H),2.36-2.27 (m, 2H), 2.18 (s, 3H), 2.05-2.00 (m, 2H), 2.00-1.95 (m, 2H),1.94-1.91 (m, 1H), 1.91-1.84 (m, 2H), 1.77-1.66 (m, 3H), 0.58 (s, 3H),0.51 (d, J=4.4 Hz, 2H), 0.39 (dd, J=2.2, 3.7 Hz, 2H). HRMS (ESI) (m/z)calc'd for C₃₁H₃₇N₂O [M+H]⁺: 453.6383. found 453.6381.

β-Cyclopropylmethylamine 40B

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-methylcyclopropylamine 40B (1.1 mg, 85%). ¹H NMR (500 MHz, CD₃OD)Shift=9.21 (s, 1H), 8.39 (d, J=5.4 Hz, 1H), 8.00-7.98 (m, 1H), 7.90 (d,J=7.8 Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.76 (dd, J=1.5, 9.3 Hz, 1H),5.78-5.74 (m, 1H), 5.29-5.25 (m, 1H), 3.26-3.24 (m, 1H), 3.23 (t, J=9.8Hz, 1H), 3.27-3.21 (m, 1H), 2.88 (t, J=1.0 Hz, 1H), 2.56-2.49 (m, 1H),2.48-2.41 (m, 2H), 2.37 (s, 3H), 2.33-2.26 (m, 1H), 2.23-2.10 (m, 3H),2.03 (d, J=7.3 Hz, 2H), 2.01-1.96 (m, 1H), 1.96-1.88 (m, 2H), 1.88-1.82(m, 1H), 1.79-1.68 (m, 2H), 0.59 (m, 5H), 0.50-0.46 (m, 2H). HRMS (ESI)(m/z) calc'd for C₃₂H₃₉N₂O [M+H]⁺: 467.6649. found 467.6645.

β-3-Methyl-3-Oxetanamine 41B and α-3-Methyl-3-Oxetanamine 41A

β-3-Methyl-3-Oxetanamine 41B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-3-methyl-3-oxetanamine 41B (4.7 mg, 81%). ¹H NMR (500 MHz, CD₃OD)Shift=9.21 (s, 1H), 8.39 (d, J=5.4 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J=8.8Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.78-7.74 (m, 1H), 5.75 (d, J=1.5 Hz,1H), 5.29-5.26 (m, 1H), 4.61 (dd, J=3.4, 5.9 Hz, 2H), 4.36 (dd, J=5.9,8.3 Hz, 2H), 3.27-3.21 (m, 1H), 3.19-3.11 (m, 1H), 2.50 (d, J=8.3 Hz,4H), 2.29 (s, 2H), 2.23-2.13 (m, 2H), 2.02-1.95 (m, 2H), 1.94-1.87 (m,3H), 1.80-1.67 (m, 4H), 1.55 (br. s., 4H), 0.59 (s, 3H). HRMS (ESI)(m/z) calc'd for C₃₂H₃₉N₂O₂ [M+H]⁺: 483.6643. found 483.6640.

β-N-Methyl-1-(3-Methyl-3-Oxetanyl)Methanamine 42B andα-N-Methyl-1-(3-Methyl-3-Oxetanyl)Methanamine 42A

β-N-Methyl-1-(3-Methyl-3-Oxetanyl)Methanamine 42B:

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5EtOAc:Hexanes:2M NH₃ solution in MeOH) to affordβ-N-methyl-1-(3-methyl-3-oxetanyl)methanamine 42B (1.5 mg, 24%). ¹H NMR(500 MHz, CD₃OD) Shift=9.22 (s, 1H), 8.39 (d, J=5.9 Hz, 1H), 7.99 (s,1H), 7.90 (d, J=8.3 Hz, 1H), 7.81 (d, J=5.9 Hz, 1H), 7.76 (dd, J=1.7,8.5 Hz, 1H), 5.75-5.72 (m, 1H), 5.31-5.27 (m, 1H), 4.57-4.51 (m, 2H),4.32 (dd, J=1.5, 5.9 Hz, 2H), 3.28-3.22 (m, 1H), 2.69 (s, 2H), 2.57-2.31(m, 5H), 2.30-2.15 (m, 4H), 2.04-1.86 (m, 5H), 1.82 (t, J=24.9 Hz, 1H),1.77-1.69 (m, 1H), 1.68-1.57 (m, 2H), 1.39 (d, J=2.9 Hz, 6H), 0.58 (s,3H). HRMS (ESI) (m/z) calc'd for C₃₄H₄₃N₂O₂ [M+H]⁺: 511.7174. found511.7173.

β-t-Butylamine 43B and α-t-Butylamine 43A

β-t-Butylamine 43B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 50:1 EtOAc:triethylamine) to afford β-t-butylamine 43B (3.4 mg,60%). ¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.49 (d, J=5.9 Hz,1H), 7.79 (s, 1H), 7.75 (d, J=8.3 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.59(d, J=8.3 Hz, 1H), 5.75 (d, J=2.0 Hz, 1H), 5.29 (m, 1H), 3.14 (dd,J=10.8, 10.8 Hz, 1H), 2.52 (dd, J=11.7, 8.8 Hz, 1H), 2.32-2.38 (m, 2H),2.13-2.26 (m, 5H), 2.01-2.08 (m, 2H), 1.94 (dd, J=17.6, 5.4 Hz, 1H),1.84-1.87 (m, 3H), 1.62-1.74 (m, 3H), 1.25 (br s, 9H). HRMS (ESI) (m/z)calc'd for C₃₂H₄₁N₂O [M+H]⁺: 469.3219. found 469.3265.

β-Aziridine 78B and α-Aziridine 78A

To a solution of ketone 13 (6 mg, 0.0146 mmol) in methanol (0.5 mL) wasadded 2-chloroethylamine hydrochloride (5.1 mg, 0.0437 mmol), followedby triethylamine (0.006 mL, 0.0437 mmol). This mixture was stirred atroom temperature for 15 minutes. Glacial acetic acid (0.0025 mL, 0.0437mmol) was added and this mixture was stirred at room temperature for 20minutes. This mixture was cooled to 0° C. and sodium cyanoborohydride(3.2 mg, 0.0510 mml) was added. The reaction was allowed to warm to roomtemperature over 16 hours and then quenched with saturated solution ofammonium chloride (5 mL). This mixture was extracted with ethyl acetate(3×8 mL). The combined organic fractions were dried over anhydrousmagnesium sulfate, filtered and concentrated. The crude was purified bysilica gel chromatography (ethylacetate, 2% triethylamine as eluent) toafford the desired β-aziridine 78B (4.5 mg, 72% yield).

¹H NMR (500 MHz, CDCl₃) δ=9.22 (s, 1H), 8.48 (d, J=5.87 Hz, 1H), 7.79(s, 1H), 7.75 (d, J=8.80 Hz, 1H), 7.63 (d, J=5.38 Hz, 1H), 7.59 (d,J=8.80 Hz, 1H), 5.74 (d, J=1.96 Hz, 1H), 5.25 (m, 1H), 3.34 (m, 2H),3.14 (dd, J=10.27, 10.27 Hz, 1H), 2.75 (m, 3H), 2.51 (dd, J=11.25, 8.31,1H), 2.44 (m, 1H), 2.29-2.37 (m, 3H), 2.12-2.27 (m, 3H), 1.81-2.08 (m,3H), 1.54-1.74 (m, 4H), 1.15 (m, 1H), 1.05 (m, 1H), 0.55 (s, 3H). HRMS(ESI) (m/z) calc'd for C₃₀H₃₅N₂O [M+H]⁺: 439.2749 found 439.2721.

β-Hydroxyproline 65B and α-Hydroxyproline 65A

Ketone 13 was reacted with hydroxyproline methyl ester under condition‘Method B’. The crude mixture was dissolved in THF:MeOH:1 M LiOH inH₂O=3:3:1 and stirred at 55° C. for 1.5 hours. The crude mixture wasroughly concentrated and pH 3.7 sodium acetate buffer was applied,followed by the extraction with chloroform three times. The crudemixture was purified by proparative TLC (eluent: 5:1 CHCl₃:MeOH) toafford β-hydroxyproline 65B (3.7 mg, 58% in 2 steps).

¹H NMR (500 MHz, CDCl₃) Shift=9.22 (br. s., 1H), 8.47 (br. s., 1H), 7.77(br. s., 1H), 7.75 (d, J=8.2 Hz, 1H), 7.63 (br. s., 1H), 7.57 (d, J=8.2Hz, 1H), 5.77 (s, 1H), 5.30 (br. s., 1H), 4.47 (br. s., 1H), 4.21-4.08(m, 1H), 4.01-3.90 (m, 0H), 3.55 (br. s., 1H), 3.19-3.11 (m, 1H), 3.12(t, J=8.8 Hz, 1H), 2.51-2.44 (m, J=10.6, 10.6 Hz, 1H), 2.44-2.37 (m,2H), 2.35 (d, J=17.6 Hz, 2H), 2.31-2.14 (m, 6H), 2.11 (dd, J=6.2, 13.2Hz, 1H), 2.06-1.96 (m, 2H), 1.92 (dd, J=4.7, 17.6 Hz, 1H), 1.87-1.68 (m,3H), 0.53 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₃H₃₉N₂O₄ [M+H]⁺:527.2904. found 527.2921.

α-Dimethylamine 14A and β-Dimethylamine 14B

The crude mixture was purified sequentially by flash chromatography(silica gel, eluent: 20:1 EtOAc:2M NH₃ solution in MeOH) to affordα-dimethylamine 14A (2.2 mg, 68%). ¹H NMR (600 MHz, C₆D₆) Shift=9.26 (s,1H), 8.56 (d, J=5.9 Hz, 1H), 7.44-7.39 (m, 1H), 7.36 (d, J=8.2 Hz, 1H),7.21-7.20 (m, 1H), 7.20 (d, J=5.9 Hz, 1H), 5.68-5.65 (m, 1H), 5.15-5.11(m, 1H), 2.72-2.66 (m, J=10.0 Hz, 1H), 2.59 (dd, J=8.8, 11.2 Hz, 1H),2.34 (tt, J=2.9, 12.1 Hz, 1H), 2.16 (td, J=3.2, 16.0 Hz, 1H), 2.09 (s,6H), 2.13-1.92 (m, 8H), 1.85 (ddd, J=5.0, 9.0, 13.6 Hz, 1H), 1.73 (dt,J=5.3, 12.3 Hz, 1H), 1.72-1.66 (m, 2H), 1.60-1.57 (m, 1H), 1.57-1.49 (m,1H), 1.20 (dq, J=4.1, 12.3 Hz, 1H), 0.40 (s, 3H). HRMS (ESI) (m/z)calc'd for C₃₀H₃₇N₂O [M+H]⁺: 441.2900. found 441.2909.

α-Monomethylamine 24A and β-Monomethylamine 24B

The crude mixture was purified by preparative TLC (silica gel, eluent:10:1 EtOAc:2M NH₃ solution in MeOH) to afford α-monomethylamine 24A (ca.1.2 mg, 37%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9Hz, 1H), 7.81 (s, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.4 Hz, 1H),7.61 (dd, J=1.0, 8.8 Hz, 1H), 5.76 (s, 1H), 5.29 (d, J=2.4 Hz, 1H), 3.16(t, J=10.0 Hz, 1H), 2.61-2.50 (m, 3H), 2.49 (s, 3H), 2.43-2.30 (m, 3H),2.30-2.15 (m, 4H), 2.13-1.99 (m, 2H), 1.95 (dd, J=5.4, 17.6 Hz, 1H),1.88 (dq, J=4.9, 11.7 Hz, 1H), 1.79-1.60 (m, 3H), 1.19 (dq, J=4.4, 12.7Hz, 1H), 0.56 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₉H₃₅N₂O [M+H]⁺:427.2744. found 427.2759.

α-Primaryamine 62A and β-Primaryamine 62B

The crude mixture was purified by preparative TLC (silica gel, eluent:25:1 EtOAc:2M NH₃ solution in MeOH) to afford α-primaryamine 62A (3.7mg, 38%) and fi-primaryamine 62B (2.5 mg, 26%).

α-Primaryamine 62A:

¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79(s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.3 Hz, 1H), 7.59 (dd,J=1.2, 8.2 Hz, 1H), 5.74 (s, 1H), 5.27 (d, J=2.9 Hz, 1H), 3.14 (t,J=10.0 Hz, 1H), 2.85 (tt, J=3.2, 11.7 Hz, 1H), 2.51 (dd, J=8.5, 11.4 Hz,1H), 2.40-2.28 (m, 3H), 2.27-2.12 (m, 4H), 2.10-1.96 (m, 3H), 1.93 (dd,J=5.3, 17.6 Hz, 1H), 1.92-1.81 (m, 2H), 1.76-1.62 (m, 2H), 1.22 (dtd,J=4.1, 11.7, 13.5 Hz, 1H), 0.53 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₈H₃₃N₂O [M+H]⁺: 413.2587. found 413.2590.

β-Primaryamine 62B:

¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79(s, 1H), 7.75 (d, J=8.2 Hz, 1H), 7.62 (d, J=5.3 Hz, 1H), 7.59 (dd,J=1.5, 8.5 Hz, 1H), 5.73 (d, J=1.2 Hz, 1H), 5.25 (d, J=2.9 Hz, 1H), 3.47(td, J=3.7, 7.9 Hz, 1H), 3.13 (t, J=10.0 Hz, 1H), 2.58 (dt, J=5.3, 14.1Hz, 1H), 2.51 (dd, J=8.5, 11.4 Hz, 1H), 2.39-2.21 (m, 5H), 2.17 (dq,J=5.3, 9.4 Hz, 1H), 2.13 (ddd, J=2.3, 4.7, 15.8 Hz, 1H), 2.10-1.99 (m,2H), 1.93 (dd, J=5.3, 17.0 Hz, 1H), 1.86 (dq, J=5.3, 12.3 Hz, 1H), 1.82(ddd, J=1.8, 4.1, 13.5 Hz, 1H), 1.78-1.66 (m, 3H), 0.54 (s, 3H). HRMS(ESI) (m/z) calc'd for C₂₈H₃₃N₂O [M+H]⁺: 413.2587. found 413.2599.

α-Morpholine 15A and β-Morpholine 15B

The crude mixture was purified by preparative TLC (silica gel, eluent:40:1 EtOAc:MeOH) to afford α-morpholine 15A (ca. 1.5 mg, 38%). ¹H NMR(600 MHz, CDCl₃) Shift=9.23 (s, 1H), 8.49 (d, J=5.4 Hz, 1H), 7.80 (s,1H), 7.76 (d, J=8.3 Hz, 1H), 7.63 (d, J=5.4 Hz, 1H), 7.60 (d, J=8.3 Hz,1H), 5.74 (s, 1H), 5.29 (d, J=2.9 Hz, 1H), 3.73 (br. s., 4H), 3.15 (t,J=10.0 Hz, 1H), 2.60 (br. s., 4H), 2.52 (dd, J=8.5, 11.5 Hz, 2H),2.46-2.29 (m, 3H), 2.29-2.13 (m, 4H), 2.12-1.99 (m, 2H), 1.94 (dd,J=5.1, 17.3 Hz, 1H), 1.94-1.81 (m, 3H), 1.73 (td, J=8.2, 12.3 Hz, 1H),1.70-1.63 (m, 1H), 1.38 (dq, J=4.4, 12.2 Hz, 1H), 0.54 (s, 3H). HRMS(ESI) (m/z) calc'd for C₃₂H₃₉N₂O₂ [M+H]⁺: 483.3006. found 483.3000.

α-Pyrrolidine 19A and β-Pyrrolidine 19B

The crude mixture was purified by preparative TLC (silica gel, eluent:20:10:3 EtOAc:Hexanes:2M NH₃ solution in MeOH) to afford α-pyrrolidine19A (2.5 mg, 55%). ¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d,J=5.9 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.2 Hz, 1H), 7.62 (d, J=5.3 Hz,1H), 7.59 (d, J=8.8 Hz, 1H), 5.72 (s, 1H), 5.27 (d, J=2.9 Hz, 1H), 3.14(t, J=10.0 Hz, 1H), 2.63 (br. s., 4H), 2.52 (dd, J=8.8, 11.2 Hz, 1H),2.42-2.29 (m, 3H), 2.28-2.15 (m, 5H), 2.12 (d, J=12.3 Hz, 1H), 2.10-2.00(m, 2H), 1.93 (dd, J=5.3, 17.0 Hz, 1H), 1.90-1.83 (m, 2H), 1.80 (br. s.,4H), 1.72 (td, J=8.8, 12.9 Hz, 1H), 1.63 (br. s., 1H), 1.37 (dq, J=3.5,11.7 Hz, 1H), 0.53 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₂H₃₉N₂O[M+H]⁺: 467.3057. found 467.3064.

α-Azetidine 18A and β-Azetidine 18B

The crude mixture was purified by preparative TLC (silica gel, eluent:1:1 EtOAc:MeOH) to afford α-azetidine 18A (ca. 1.5 mg, 38%). ¹H NMR (500MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.4 Hz, 1H), 7.80 (s, 1H),7.77 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60 (d, J=8.8 Hz, 1H),5.74 (s, 1H), 5.28 (br. s., 1H), 3.24 (br. s., 4H), 3.16 (t, J=9.8 Hz,1H), 2.54 (dd, J=8.8, 11.2 Hz, 1H), 2.42-2.30 (m, 3H), 2.30-2.13 (m,5H), 2.12-2.00 (m, 2H), 1.95 (dd, J=5.4, 18.1 Hz, 1H), 1.93-1.78 (m,3H), 1.74 (td, J=8.3, 12.2 Hz, 1H), 1.67-1.54 (m, 3H), 1.11 (q, J=12.2Hz, 1H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₁H₃₇N₂O [M+H]⁺:453.2906. found 453.2900.

α-t-Butylamine 43A and β-t-Butylamine 43B

The crude mixture was purified by flash chromatography (silica gel,eluent: 10:1 CHCl₃:i-PrOH) to afford α-t-butylamine 43A (2.4 mg, 42%)and β-t-butylamine 43B (1.6 mg, 28%).

α-t-Butylamine 43A:

¹H NMR (500 MHz, CDCl₃) Shift=9.22 (br. s., 1H), 8.48 (d, J=5.9 Hz, 1H),7.78 (s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.3 Hz, 1H), 7.58 (dd,J=1.2, 8.2 Hz, 1H), 5.73 (s, 1H), 5.28 (d, J=2.3 Hz, 1H), 3.14 (t, J=9.7Hz, 1H), 2.49 (dd, J=8.8, 11.2 Hz, 1H), 2.46-2.40 (m, 1H), 2.39-2.29 (m,3H), 2.28-2.12 (m, 5H), 2.08-1.99 (m, 1H), 1.93 (dd, J=5.3, 17.0 Hz,1H), 1.83 (dq, J=5.0, 12.2 Hz, 2H), 1.76-1.60 (m, 3H), 1.55 (br. s.,9H), 1.26-1.18 (m, 1H), 0.54 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₂H₄₁N₂O [M+H]⁺: 469.3213. found 469.3223.

β-t-Butylamine 43B:

¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.3 Hz, 1H), 7.78(s, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.62 (d, J=5.9 Hz, 1H), 7.59 (d, J=9.4Hz, 1H), 5.73 (br. s., 1H), 5.25 (br. s., 1H), 3.38-3.23 (m, 1H), 3.13(t, J=10.0 Hz, 1H), 2.57-2.48 (m, 1H), 2.49 (dd, J=8.5, 10.9 Hz, 1H),2.40-2.27 (m, 2H), 2.25-2.08 (m, 4H), 2.07-1.98 (m, 2H), 1.93 (dd,J=5.3, 17.6 Hz, 2H), 1.84 (dq, J=5.3, 12.3 Hz, 1H), 1.76-1.66 (m, 2H),1.65-1.47 (m, 3H), 1.42-0.94 (br. s., 9H), 0.54 (s, 3H). HRMS (ESI)(m/z) calc'd for C₃₂H₄₁N₂O [M+H]⁺: 469.3213. found 469.3225.

α-Hydroxyazetidine 70A and β-Hydroxyazetidine 70B

The crude mixture was purified by preparative TLC (silica gel, eluent:2:3 EtOAc:MeOH) to afford α-hydroxyazetidine 70A (1.2 mg, 30%). ¹H NMR(500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.4 Hz, 1H), 7.80 (s,1H), 7.77 (d, J=8.3 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60 (d, J=8.3 Hz,1H), 5.77 (s, 1H), 5.32 (d, J=2.4 Hz, 1H), 4.66-4.55 (m, 1H), 3.94 (br.s., 2H), 3.34 (br. s., 2H), 3.16 (t, J=9.8 Hz, 1H), 2.53 (dd, J=8.5,11.5 Hz, 1H), 2.41 (dd, J=15.6, 28.3 Hz, 2H), 2.34 (dt, J=4.9, 11.2 Hz,1H), 2.28 (t, J=11.2 Hz, 1H), 2.25-2.14 (m, 3H), 2.10-1.98 (m, 2H), 1.95(dd, J=5.4, 17.6 Hz, 1H), 1.96-1.89 (m, 1H), 1.86 (dd, J=5.4, 12.2 Hz,1H), 1.84-1.76 (m, 2H), 1.74 (td, J=8.4, 12.4 Hz, 1H), 1.65 (dt, J=7.8,10.5 Hz, 1H), 1.40-1.27 (m, 1H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'dfor C₃₁H₃₇N₂O₂ [M+H]⁺: 469.2850. found 469.2872.

α-Hydroxymethylazetidine 69A and β-Hydroxymethylazetidine 69B

The crude mixture was purified by preparative TLC (silica gel, eluent:1:1 EtOAc:MeOH) to afford α-hydroxymethylazetidine 69A (2.3 mg, 53%). ¹HNMR (600 MHz, CDCl₃) Shift=9.23 (s, 1H), 8.49 (d, J=5.9 Hz, 1H), 7.80(s, 1H), 7.76 (d, J=8.2 Hz, 1H), 7.63 (d, J=5.9 Hz, 1H), 7.59 (d, J=8.8Hz, 1H), 5.76 (s, 1H), 5.30 (d, J=2.9 Hz, 1H), 3.65-3.35 (m, 4H), 3.15(t, J=10.0 Hz, 1H), 2.52 (dd, J=8.5, 11.4 Hz, 1H), 2.40 (dd, J=16.4,25.8 Hz, 2H), 2.33 (dt, J=4.1, 11.7 Hz, 1H), 2.26 (t, J=11.4 Hz, 1H),2.24 (m, 3H), 2.09-2.00 (m, 1H), 1.98 (br. s., 1H), 1.94 (dd, J=5.0,17.3 Hz, 1H), 1.92-1.77 (m, 4H), 1.73 (td, J=8.2, 12.9 Hz, 1H),1.67-1.59 (m, 1H), 1.58-1.50 (br. s., 3H), 1.39-1.28 (m, 1H), 0.58-0.51(s, 3H). HRMS (ESI) (m/z) calc'd for C₃₂H₃₉N₂O₂ [M+H]⁺: 483.3006. found483.3000.

α-Aminoethylsulfonamide 71A and β-Aminoethylsulfonamide 71B

The crude mixture was purified by preparative TLC (silica gel, eluent:100% EtOAc) to afford β-aminoethylsulfonamide 71B (0.7 mg, 15%) andα-aminoethylsulfonamide 71A (1.1 mg, 24%).

β-Aminoethylsulfonamide 71B:

¹H NMR (500 MHz, CD₃OD) Shift=9.21-9.17 (m, 1H), 8.37 (d, J=5.9 Hz, 1H),7.97 (s, 1H), 7.88 (d, J=8.3 Hz, 1H), 7.79 (d, J=5.4 Hz, 1H), 7.74 (dd,J=1.5, 8.8 Hz, 1H), 5.74-5.69 (m, 1H), 5.28-5.22 (m, 1H), 3.26-3.18 (m,J=9.8 Hz, 1H), 3.14-3.00 (m, 4H), 2.59-2.38 (m, 4H), 2.37-2.26 (m, 2H),2.23-2.06 (m, 3H), 2.03-1.86 (m, 5H), 1.85-1.76 (m, 1H), 1.76-1.59 (m,3H), 0.57 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₈N₃O₃S [M+H]⁺:520.2628. found 520.2640.

α-Aminoethylsulfonamide 71A:

¹H NMR (500 MHz, CD₃OD) Shift=9.19 (s, 1H), 8.37 (d, J=5.9 Hz, 1H), 7.97(s, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.79 (d, J=5.9 Hz, 1H), 7.74 (dd,J=1.7, 8.6 Hz, 1H), 5.77-5.71 (m, 1H), 5.30-5.24 (m, 1H), 3.29-3.24 (m,2H), 3.23 (dd, J=9.0, 11.0 Hz, 1H), 3.13 (dt, J=2.7, 6.7 Hz, 2H), 2.73(tt, J=3.1, 12.0 Hz, 1H), 2.50 (dd, J=8.6, 11.5 Hz, 1H), 2.47-2.38 (m,2H), 2.39-2.34 (m, 1H), 2.32 (d, J=11.2 Hz, 1H), 2.30-2.22 (m, 2H), 2.17(dtd, J=5.9, 9.3, 14.7 Hz, 2H), 2.10-2.03 (m, 1H), 2.01 (s, 1H),2.00-1.92 (m, 1H), 1.90 (dd, J=6.4, 17.1 Hz, 1H), 1.72 (td, J=8.3, 12.3Hz, 1H), 1.68-1.60 (m, 2H), 1.18 (dq, J=4.4, 12.2 Hz, 1H), 0.56 (s, 3H).HRMS (ESI) (m/z) calc'd for C₃₀H₃₈N₃O₃S [M+H]⁺: 520.2628. found520.2643.

α-Hydroxyaminomethyloxetane 72A and β-Hydroxyaminomethyloxetane 72B

The crude mixture was purified by preparative TLC (silica gel, eluent:100% EtOAc) to afford α-hydroxyaminomethyloxetane 72A (1.5 mg, 34%). ¹HNMR (500 MHz, CD₃OD) Shift=9.19 (s, 1H), 8.38 (d, J=5.4 Hz, 1H), 7.97(s, 1H), 7.88 (d, J=8.3 Hz, 1H), 7.79 (d, J=5.9 Hz, 1H), 7.74 (dd,J=1.5, 8.8 Hz, 1H), 5.84-5.78 (m, 1H), 5.35-5.30 (m, 1H), 4.64 (d, J=7.3Hz, 2H), 4.57 (dd, J=3.7, 7.1 Hz, 2H), 3.48-3.40 (m, 2H), 3.24 (dd,J=9.0, 11.0 Hz, 2H), 2.54-2.48 (m, J=8.8 Hz, 1H), 2.48-2.40 (m, 3H),2.40-2.24 (m, 4H), 2.24-2.13 (m, 3H), 2.01-1.85 (m, 4H), 1.80-1.64 (m,2H), 1.46 (dq, J=4.9, 12.2 Hz, 1H), 0.57 (s, 3H). HRMS (ESI) (m/z)calc'd for C₃₂H₃₉N₂O₃ [M+H]⁺: 499.2955. found 499.2933.

β-PEGamine 75B and α-PEGamine 75A

β-PEGamine 75B:

The crude mixture was purified by preparative TLC (silica gel, eluent:5:5:1 EtOAc:Dichloromethane:2M NH₃ solution in MeOH) to affordβ-PEGamine 75B (1.0 mg, 18%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s,1H), 8.50 (d, J=5.9 Hz, 1H), 7.81 (s, 1H), 7.77 (d, J=8.3 Hz, 1H), 7.64(d, J=5.9 Hz, 1H), 7.61 (dd, J=1.7, 8.5 Hz, 1H), 5.72 (d, J=1.5 Hz, 2H),5.25 (dd, J=2.2, 5.1 Hz, 1H), 3.71-3.64 (m, 6H), 3.62 (t, J=5.4 Hz, 2H),3.58 (dd, J=3.7, 5.6 Hz, 2H), 3.41 (s, 3H), 3.19-3.12 (m, J=10.7 Hz,1H), 3.11 (t, J=3.9 Hz, 1H), 2.79 (t, J=5.4 Hz, 2H), 2.56 (t, J=16.1 Hz,1H), 2.52 (dd, J=8.3, 11.2 Hz, 1H), 2.42-2.29 (m, 3H), 2.27-2.13 (m,2H), 2.12-2.01 (m, 2H), 2.02 (dd, J=3.7, 13.9 Hz, 1H), 1.95 (br. s.,2H), 1.86 (dq, J=5.4, 12.2 Hz, 1H), 1.80-1.73 (m, 1H), 1.71 (dd, J=3.2,11.5 Hz, 1H), 1.68-1.58 (m, 2H), 0.56 (s, 3H). HRMS (ESI) (m/z) calc'dfor C₃₅H₄₇N₂O₄ [M+H]⁺: 559.3530. found 559.3545.

α-PEGamine 75A:

The crude mixture was purified by preparative TLC (silica gel, eluent:100:5:1 EtOAc:MeOH:Triethylamine) to afford α-PEGamine 75A (1.1 mg,20%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz,1H), 7.81 (s, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.61(dd, J=1.5, 8.8 Hz, 1H), 5.75 (d, J=2.0 Hz, 1H), 5.28 (dd, J=2.2, 5.1Hz, 1H), 3.70-3.65 (m, 7H), 3.64 (t, J=5.4 Hz, 2H), 3.61-3.55 (m, 2H),3.41 (s, 3H), 3.16 (dd, J=9.0, 10.5 Hz, 1H), 2.87 (t, J=5.1 Hz, 2H),2.67 (t, J=11.5 Hz, 1H), 2.54 (dd, J=8.3, 11.7 Hz, 1H), 2.38 (d, J=16.1Hz, 3H), 2.24 (d, J=12.2 Hz, 2H), 2.22-2.14 (m, 2H), 2.11-2.02 (m, 2H),1.95 (dd, J=5.4, 16.6 Hz, 1H), 1.88 (dq, J=5.4, 12.2 Hz, 1H), 1.78-1.69(m, 2H), 1.68-1.62 (m, 1H), 1.27-1.19 (m, 1H), 0.55 (s, 3H). HRMS (ESI)(m/z) calc'd for C₃₅H₄₇N₂O₄ [M+H]⁺: 559.3530. found 559.3542.

α-Methylsulfonamide 73A

The crude mixture was purified by preparative TLC (silica gel, eluent:40:1 MeOH:Dichloromethane) to afford α-methylsulfonamide 73A (2.0 mg,82%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (br. s., 1H), 8.51 (d, J=4.9Hz, 1H), 7.80 (s, 1H), 7.77 (d, J=8.3 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H),7.60 (dd, J=1.5, 8.3 Hz, 1H), 5.78 (d, J=1.5 Hz, 1H), 5.36-5.29 (m, 1H),4.24 (d, J=7.8 Hz, 1H), 3.53 (tdt, J=3.9, 7.7, 11.7 Hz, 1H), 3.20-3.11(m, J=10.7 Hz, 1H), 3.04 (s, 3H), 2.51 (dd, J=8.3, 11.7 Hz, 1H), 2.37(d, J=16.6 Hz, 3H), 2.28 (br. s., 2H), 2.25-2.10 (m, 4H), 2.10-2.00 (m,1H), 1.96 (dd, J=5.4, 17.6 Hz, 1H), 1.88 (dt, J=5.4, 11.7 Hz, 1H), 1.85(t, J=12.2 Hz, 1H), 1.80-1.66 (m, 2H), 1.47-1.35 (m, J=4.4 Hz, 1H), 0.55(s, 3H); HRMS (ESI) (m/z) calc'd for C₂₉H₃₅N₂O₃S [M+H]⁺: 491.2363. found491.2387.

β-Methylsulfonamide 73B

The crude mixture was purified by preparative TLC (silica gel, eluent:40:1 MeOH:Dichloromethane) to afford β-methylsulfonamide 73B (1.7 mg,90%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz,1H), 7.80 (s, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60(dd, J=1.5, 8.3 Hz, 1H), 5.79 (d, J=1.5 Hz, 1H), 5.35-5.30 (m, 1H), 4.31(d, J=6.3 Hz, 1H), 4.00 (quind, J=3.6, 6.7 Hz, 1H), 3.16 (dd, J=9.0,10.5 Hz, 1H), 3.03 (s, 3H), 2.52 (dd, J=8.5, 11.5 Hz, 1H), 2.41 (t,J=16.1 Hz, 1H), 2.39 (br. s., 2H), 2.32-2.26 (m, 2H), 2.26-2.14 (m, 3H),2.12-1.99 (m, 2H), 1.96 (dd, J=5.1, 17.3 Hz, 2H), 1.86 (dq, J=5.4, 12.2Hz, 1H), 1.83-1.72 (m, 3H), 0.56 (s, 3H); HRMS (ESI) (m/z) calc'd forC₂₉H₃₅N₂O₃S [M+H]⁺: 491.2363. found 491.2376.

α-Methyl-Methylsulfonamide 76A

The crude mixture was purified by preparative TLC (silica gel, eluent:40:1 MeOH:Dichloromethane) to afford α-methyl-methylsulfonamide 76A (0.7mg, 57%). ¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.51 (d, J=5.9 Hz,1H), 7.81 (s, 1H), 7.78 (d, J=8.3 Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60(dd, J=1.5, 8.3 Hz, 1H), 5.79 (s, 1H), 5.33 (dd, J=2.2, 5.6 Hz, 1H),3.98 (tt, J=3.4, 12.4 Hz, 1H), 3.16 (dd, J=9.0, 10.5 Hz, 1H), 2.89 (s,3H), 2.80 (s, 3H), 2.52 (dd, J=8.5, 11.5 Hz, 1H), 2.44 (ddd, J=2.7, 4.1,16.6 Hz, 1H), 2.36 (br. s., 3H), 2.28 (d, J=10.7 Hz, 2H), 2.21 (dq,J=4.9, 9.1 Hz, 1H), 2.14 (t, J=12.7 Hz, 1H), 2.10-2.00 (m, 0H), 1.97(dd, J=5.4, 17.6 Hz, 1H), 1.93 (dd, J=2.9, 12.2 Hz, 1H), 1.88 (dd,J=5.4, 12.2 Hz, 1H), 1.87-1.81 (m, 1H), 1.80-1.60 (m, 3H), 0.56 (s, 3H).HRMS (ESI) (m/z) calc'd for C₃₀H₃₇N₂O₃S [M+H]⁺: 505.2519. found505.2537.

Example 2 Another Possible Route from Isoquinoline Compound 10 to 12

A new route to isoquinoline 12 was designed. See Scheme 2-1 below.Triflation/Suzuki cross-coupling reaction was achieved on a similarsubstrate with the designated reagents shown in the figure. See, e.g.,Nicolaou et al. J. Am. Chem. Soc. 2009, 131, 10587-10597.

Synthesis of Triflate 20

To a solution of monoketone 10 (200 mg, 584 μmol, 1.00 equiv) in THF (4mL) was added NaHMDS (1 M, 701 μL, 701 μmol, 1.20 equiv) at −78° C.dropwise. After stirring 1.5 h, PhNTf₂ (313 mg, 876 μmol, 1.50 equiv) inTHF (2.5 mL) was cannulated and the reaction mixture was warmed up to 0°C. After additional 30 min, saturated NH₄Cl solution (8 mL) was added tothe stirred reaction mixture and diluted with EtOAc (10 mL). The layerswere separated and the aqueous layer was extracted with EtOAc (2×6 mL)and the organic layers were combined, washed with brine (15 mL), driedover Na₂SO₄, and concentrated under reduced pressure. The resultingresidue was then purified by flash column chromatography (silica gel,eluent: 8:1→5:1 Hexanes:EtOAc) to provide triflate 20 (237 mg, 86%). ¹HNMR (500 MHz, CDCl₃) Shift=5.76 (s, 1H), 5.67 (br. s., 1H), 5.32 (dd,J=2.0, 4.9 Hz, 1H), 4.02-3.94 (m, 4H), 2.67 (dd, J=6.8, 10.7 Hz, 1H),2.49 (t, J=14.6 Hz, 1H), 2.45 (ddd, J=3.7, 6.5, 15.2 Hz, 1H), 2.38-2.28(m, 4H), 2.17 (ddd, J=1.5, 10.7, 12.7 Hz, 1H), 2.12 (d, J=13.2 Hz, 1H),2.10 (dd, J=5.9, 17.6 Hz, 1H), 1.98 (dd, J=2.7, 13.4 Hz, 1H), 1.88 (ddd,J=7.6, 8.9, 12.8 Hz, 1H), 1.80 (tdd, J=2.4, 4.8, 12.7 Hz, 1H), 1.74-1.63(m, 2H), 1.03 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₂H₂₆O₆F₃S [M+H]⁺:475.1397. found 475.1411.

Synthesis of Suzuki Cross-Coupling for 17,18-Unsaturated Isoquinoline 21from Triflate 20

To a solution of triflate 20 (1.00 equiv) and isoquinoline-7-boronicacid (3.00 equiv) in 1,4-dioxane and H₂O (10:1, 0.02M) was added K₂CO₃(3.00 equiv) and the solution was bubbled through inert Ar for 5 min.Pd(dppf)Cl₂.CH₂Cl₂ (0.05 equiv) was added and the reaction mixture wasstirred at 80° C. for 1 h. The mixture was allowed to cool to roomtemperature and saturated NaHCO₃ solution was applied. The mixture wasdiluted with EtOAc and the layers were separated. The aqueous layer wasextracted with EtOAc and the combined organic layers were washed withbrine dried over Na₂SO₄, and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silicagel, eluent: 2:1→1:1→1:2 Hexanes:EtOAc) to provide 17,18-unsaturatedisoquinoline 21 (490 mg, 84%). ¹H NMR (500 MHz, CDCl₃) Shift=9.23 (s,1H), 8.49 (d, J=5.4 Hz, 1H), 7.94 (s, 1H), 7.85-7.81 (m, 1H), 7.80-7.75(m, 1H), 7.63 (d, J=5.4 Hz, 1H), 6.26 (br. s., 1H), 5.82 (s, 1H), 5.40(d, J=3.4 Hz, 1H), 4.08-3.90 (m, 4H), 2.76 (dd, J=7.1, 11.0 Hz, 1H),2.58 (dt, J=5.4, 17.6 Hz, 1H), 2.56-2.40 (m, 3H), 2.40-2.28 (m, 4H),2.16 (d, J=13.2 Hz, 1H), 2.02 (dd, J=2.0, 13.2 Hz, 1H), 1.94 (td, J=8.8,13.2 Hz, 1H), 1.81 (td, J=2.0, 12.7 Hz, 1H), 1.76-1.67 (m, 2H), 1.18 (s,3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₂NO₃ [M+H]⁺: 454.2377. found454.2366.

Synthesis of Isoquinoline 12 from 17,18-Unsaturated Isoquinoline 21

To a solution of 17,18-unsaturated isoquinoline 21 (400 mg, 877 μmol,1.0 equiv) in THF (36 mL) was added 10 wt % Pd/C (280 mg, 263 μmol, 0.30equiv) and H₂ balloon was installed. After 3 h, the reaction mixture wasfiltered through a pad of celite and washed with 0.2 M NH₃ solution inMeOH (40 mL), concentrated under reduced pressure. The residue waspurified by flash column chromatography (silica gel, eluent: 1:1-1:2Hexanes:EtOAc) to provide isoquinoline 12 (325 mg, 80%). Spectral datawas consistent with isoquinoline 12 constructed from1-chloroisoquinoline adduct 11.

Example 3 Synthesis of Isoquinoline Analogs

Suzuki Cross-Coupling for 17,18-Unsaturated 5-Indazole 56 from Triflate20

To a solution of triflate 20 (1.00 equiv) and indazole-5-boronic ester(3.00 equiv) in 1,4-dioxane and H₂O (10:1, 0.02M) was added K₂CO₃ (3.00equiv) and the solution was bubbled through inert Ar for 5 min.Pd(dppf)Cl₂.CH₂Cl₂ (0.05 equiv) was added and the reaction mixture wasstirred at 80° C. for 1 h. The mixture was allowed to cool to roomtemperature and saturated NaHCO₃ solution was applied. The mixture wasdiluted with EtOAc and the layers were separated. The aqueous layer wasextracted with EtOAc and the combined organic layers were washed withbrine dried over Na₂SO₄, and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silicagel, eluent: 1:3→1:1 EtOAc:Hexanes) to afford 17,18-unsaturated6-indazole 56 (13 mg, 70%). ¹H NMR (500 MHz, CDCl₃) Shift=8.04 (s, 1H),7.67 (d, J=10.0 Hz, 1H), 7.49 (s, 1H), 7.27 (d, J=10.0 Hz, 1H), 6.12(br. s., 1H), 5.78 (br. s., 1H), 5.36 (t, J=5.0 Hz, 1H), 3.99 (m, 4H),2.72 (m, 1H), 2.53-2.44 (m, 3H), 2.40 (br. d., J=10.0 Hz, 1H), 2.36-2.25(m, 4H), 2.14 (d, J=10.0 Hz, 1H), 2.00 (dd, J=10.0, 5.0 Hz, 1H),1.93-1.87 (m, 1H), 1.79 (m, 1H), 1.72-1.66 (m, 2H), 1.10 (s, 3H). HRMS(ESI) (m/z) calc'd for C₂₈H₃₁N₂O₃ [M+H]⁺: 443.5573. found 443.5571.

Suzuki Cross-Coupling for 17,18-Unsaturated 6-Indazole 59 from Triflate20

To a solution of triflate 20 (1.00 equiv) and indazole-6-boronic ester(3.00 equiv) in 1,4-dioxane and H₂O (10:1, 0.02M) was added K₂CO₃ (3.00equiv) and the solution was bubbled through inert Ar for 5 min.Pd(dppf)Cl₂.CH₂Cl₂ (0.05 equiv) was added and the reaction mixture wasstirred at 80° C. for 1 h. The mixture was allowed to cool to roomtemperature and saturated NaHCO₃ solution was applied. The mixture wasdiluted with EtOAc and the layers were separated. The aqueous layer wasextracted with EtOAc and the combined organic layers were washed withbrine dried over Na₂SO₄, and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silicagel, eluent: 1:4→3:1 EtOAc:Hexanes) to afford 17,18-unsaturated5-indazole 59 (5.9 mg, 65%). ¹H NMR (500 MHz, CDCl₃) Shift=10.04 (br s,1H), 8.05 (s, 1H), 7.75 (s, 1H), 7.46 (ABq, J_(AB)=8.8 Hz, Δν=33.5 Hz,2H), 6.02 (s, 1H), 5.79 (s, 1H), 5.38 (dd, J=3.4, 3.4 Hz, 1H), 3.94-4.01(m, 4H), 2.73 (dd, J=10.7, 6.8 Hz, 1H), 2.44-2.53 (m, 3H), 2.40 (d, 12.2Hz, 1H), 2.28-2.36 (m, 3H), 2.15 (d, J=13.2 Hz, 1H), 2.03 (par obs d,J=11.2 Hz, 1H), 2.01 (par obs dd, J=13.2, 2.4 Hz, 1H), 1.91 (dt,J=12.21, 9.3 Hz, 1H), 1.77-1.82 (m, 1H), 1.66-1.73 (m, 2H), 1.10 (s,3H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₁N₂O₃ [M+H]⁺: 443.2335. found443.4956.

β-Dimethylamine Aminomethylpyridine 46B and α-DimethylamineAminomethylpyridine 46A

β-Dimethylamine Aminomethylpyridine 46B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 10:1 EtOAc:2M NH₃ solution in MeOH) to afford β-dimethylamineaminomethylpyridine 46B (5 mg, 55%). ¹H NMR (500 MHz, CDCl₃) Shift=8.58(s, 1H), 8.51 (dd, J=1.5, 4.9 Hz, 1H), 7.71 (td, J=2.0, 7.8 Hz, 1H),7.26 (dd, J=4.9, 7.8 Hz, 1H), 5.71 (s, 1H), 5.24 (dd, J=2.4, 4.4 Hz,1H), 3.88-3.80 (m, 2H), 2.81 (t, J=9.0 Hz, 1H), 2.45 (dt, J=6.3, 13.7Hz, 1H), 2.46-2.37 (m, 1H), 2.30 (br. s., 6H), 2.26-2.19 (m, 3H),2.17-2.07 (m, 5H), 1.92 (dd, J=2.9, 13.7 Hz, 2H), 1.79 (dddd, J=4.9,8.3, 10.3, 13.2 Hz, 1H), 1.74-1.59 (m, 4H), 1.39 (ddt, J=4.4, 9.8, 12.7Hz, 1H), 0.80 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₇H₃₈N₃O [M+H]⁺:420.3009. found 420.2999.

β-Dimethylamine 17,18-Unsaturated Amidepyridine 49B and α-Dimethylamine17,18-Unsaturated Amidepyridine 49A

β-Dimethylamine 17,18-Unsaturated Amidepyridine 49B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 8:1 EtOAc:2M NH₃ solution in MeOH) to afford β-dimethylamine17,18-unsaturated amidepyridine 49B (2.1 mg, 44%). ¹H NMR (500 MHz,CDCl₃) Shift=8.58 (d, J=2.4 Hz, 1H), 8.36 (dd, J=1.0, 4.9 Hz, 1H), 8.23(td, J=2.0, 8.3 Hz, 1H), 7.49 (s, 1H), 7.29 (dd, J=4.9, 8.8 Hz, 1H),6.57 (br. s., 1H), 5.73 (s, 1H), 5.33 (dd, J=2.0, 5.4 Hz, 1H), 2.66-2.56(m, 2H), 2.54 (dd, J=3.2, 6.6 Hz, 1H), 2.51-2.32 (m, 5H), 2.28 (br. s.,6H), 2.20 (ddd, J=1.2, 11.0, 12.7 Hz, 1H), 2.10 (td, J=6.4, 13.1 Hz,2H), 1.97-1.89 (m, 3H), 1.76 (dt, J=7.8, 11.2 Hz, 1H), 1.66-1.55 (m,1H), 1.14 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₇H₃₄N₃O₂ [M+H]⁺:432.2646. found 432.2649.

β-Dimethylamine 17,18-Unsaturated Phthalazine 55B and α-Dimethylamine17,18-Unsaturated Phthalazine 55A

β-Dimethylamine 17,18-unsaturated phthalazine 55B: The crude mixture waspurified by flash chromatography (silica gel, eluent: 9:1 EtOAc:2M NH₃solution in MeOH) to afford β-dimethylamine 17,18-unsaturatedphthalazine 55B (5.5 mg, 73%). ¹H NMR (500 MHz, CDCl₃) Shift=9.50 (s,2H), 7.97-8.03 (m, 1H), 7.89 (d, J=4.39 Hz, 2H), 6.34-6.39 (m, 1H),5.77-5.83 (m, 1H), 5.32-5.43 (m, 1H), 2.72 (dd, J=11.23, 6.84 Hz, 1H),2.38-2.50 (m, 4H), 2.47 (br. m., 6H), 2.24-2.30 (m, 3H), 2.09-2.20 (m,2H), 2.03 (d, J=10.25 Hz, 2H), 1.88-1.99 (m, 2H), 1.75-1.86 (m, 2H),1.17 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₃N₃O [M+H]⁺: 440.5998.found 440.5995.

β-Dimethylamine 17,18-unsaturated 6-indazole 58B and α-Dimethylamine17,18-unsaturated 6-indazole 58A

β-Dimethylamine 17,18-Unsaturated 6-Indazole 58B:

The crude mixture was purified by flash chromatography (silica gel,eluent: 9:1 EtOAc 2M NH₃ solution in MeOH) to afford β-dimethylamine17,18-unsaturated 6-indazole 58B (3.9 mg, 73%). ¹H NMR (500 MHz, CDCl₃)Shift=8.03 (s, 1H), 7.67 (d, J=8.30 Hz, 1H), 7.49 (s, 1H), 7.25-7.28 (=,1H), 6.12 (t, J=2.44 Hz, 1H), 5.75 (s, 1H), 5.33 (t, J=3.42 Hz, 1H),3.22-3.38 (Hz, 1H), 2.71 (dd, J=11.23, 6.84 Hz, 1H), 2.44-2.50 (m, 4H),2.41 (br. m., 6H), 2.226-2.31 (m, 3H), 2.09-112.20 (m, 2H), 2H), 2.04(d, J=2.93 Hz, 2H), 1.78 (td, J=11.35, 7.08 Hz, 2H), 1.52-1.64 (m, 1H),1.07-1.15 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₃N₃O [M+H]⁺:428.5891. found 428.5889.

β-Dimethylamine 17,18-Unsaturated 5-Indazole 61B and α-Dimethylamine17,18-Unsaturated 5-Indazole 61A

β-Dimethylamine 17,18-Unsaturated 5-Indazole 61B:

The crude mixture was purified by flash column chromatography (silicagel, eluent: 9:1 EtOAc:2M NH₃ solution in MeOH) to affordβ-dimethylamine 17,18-unsaturated 5-indazole 61B (2.5 mg, 70%). ¹H NMR(500 MHz, CDCl₃) Shift=8.05 (s, 1H), 7.75 (s, 1H), 7.47 (ABq, J_(AB)=8.8Hz, Δν=33.5 Hz, 2H), 6.02 (dd, J=2.0, 2.0 Hz, 1H), 5.74 (s, 1H), 5.33(dd, J=3.4, 3.4 Hz, 1H), 2.72 (dd, J=11.2, 6.8 Hz, 1H), 2.43-2.48 (m,5H), 2.37-2.41 (m, 2H), 2.26-2.35 (m, 8H), 2.11 (m, 2H), 2.04 (d, J=6.8Hz, 1H), 1.88-1.98 (m, 3H), 1.77 (m, 1H), 1.10 (s, 3H). HRMS (ESI) (m/z)calc'd for C₂₈H₃₄N₃O [M+H]⁺: 428.2702. found 428.2653.

Synthesis of β-Dimethylamine Amidepyridine 50B

To a solution off β-dimethylamine 17,18-unsaturated amidepyridine 49B(ca. 1.2 mg) in MeOH (300 μL) was added Mg (ca. 1 mg) and stirred atroom temperature for 48 h. The reaction mixture was added H₂O (700 μL)and diluted with EtOAc (700 μL). The aqueous phase was extracted withEtOAc (2×0.5 mL) and the combined organic phases were washed with brine(0.5 mL), dried over Na₂SO₄, and concentrated under reduced pressure.The residue was purified by HPLC (Eclipse XDB-C8 column, 9.4 mm×25 cm;gradient=0%-35% MeCN (0.1% formic acid):H₂O (0.1% formic acid) over 30min) to provide β-dimethylamine amidepyridine 50B (ca. 0.3 mg, 25%). Duetoo the small quantity, only diagnostic peaks were assigned. ¹H NMR (500MHz, CDCl₃) Shift=8.56 (br. s, 1H), 8.37 (d, J=3.4 Hz, 1H), 8.21 (d,J=8.3 Hz, 1H), 7.09 (br. s., 1H), 5.81 (s, 1H), 5.39-5.33 (m, 1H), 2.78(br. s., 6H), 0.83 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₇H₃₅N₃O₂[M+H]⁺: 434.2802. found 434.2815.

Synthesis of Phthalazine 6-Triflate 51

To a solution of 6-phthalazinol (588.4 mg, 4.26 mmol, 1.0 equiv) inCHCl₃ was added N-Phenyl-bis(trifluoromethanesulfonimide) (1.73 g, 4.83mmol, 1.2 equiv), Et₃N (0.9 mL, 6.04 mmol, 1.5 equiv) and DMAP (cat.).The mixture was warmed to 60° C. and stirred for 3 h. The reaction wascooled to room temperature and quenched with sat. NaHCO₃ and CH₂Cl₂ andthe layers were separated. The aqueous layer was extracted with CH₂Cl₂.The organic layers were combined dried over Na₂SO₄ and concentratedunder reduced pressure. The crude mixture was purified by flash columnchromatography (silica gel, eluent: 9:1 Dichloromethane:MeOH) to affordphthalazine 6-triflate 51 (995 mg, 90%). ¹H NMR (500 MHz, CDCl₃)Shift=9.68 (d, J=3.91 Hz, 2H) 8.19 (d, J=8.79 Hz, 1H) 7.96 (br. s., 1H)7.84-7.89 (m, 1H). HRMS (ESI) (m/z) calc'd for C₉H₆F₃N₂O₃S [M+H]⁺:279.2157. found 279.2152.

Synthesis of Phthalazine 6-Trimethyltin 52

To a solution of phthalazine 6-triflate 52 (992 mg, 3.57 mmol, 1.0equiv) in C₆H₆ was added LiCl (907 mg, 21.59 mmol, 6.0 equiv), Pd(PPh₃)₄(412 mg, 0.3565 mmol, 0.1 equiv) and (Me₃Sn)₂ (0.78 mL, 3.743 mmol, 1.05equiv). The solution was bubbled with argon in a sonicator for 10 minsand the mixture was warmed to 105° C. and stirred for 1 h. The reactionwas cooled to rt, diluted with ethyl acetate and filtered over celite.The organic portion was washed with sat NaHCO₃ and dried over Na₂SO₄ andthen concentrated under reduced pressure. The crude mixture was purifiedby flash column chromatography (silica gel, eluent: 1:1 EtOAc:Hexanes)to afford phthalazine 6-trimethyltin 52 (656 mg, 63%). ¹H NMR (500 MHz,CDCl₃) Shift=9.54 (d, J=4.39 Hz, 2H) 8.12 (br. s., 1H) 8.08 (d, J=7.81Hz, 1H) 7.92 (d, J=7.81 Hz, 1H) 0.43 (s, 9H). HRMS (ESI) (m/z) calc'dfor C₁₁H₁₅N₂Sn [M+H]⁺: 293.9602, found 293.9601.

Synthesis of 17,18-Unsaturated Phthalazine 53

To a solution of triflate 20 (20 mg, 42.15 μmol, 1.0 equiv) in DMSO wasadded (trimethylstannyl)phthalazine 52 (31 mg, 105.40 μmol, 2.0 equiv),CuCl (42 mg, 421.50 μmol, 10.0 equiv) and LiCl (18 mg, 421.50 μmol, 10.0equiv). The mixture was deoxygenated by freeze-thaw method four timesand Pd(PPh₃) (5 mg, 4.22 μmol, 0.1 equiv) was added. The mixture washeated to 60° C. and stir 1 h. The reaction was quenched with 5% NH₄OHand ethyl acetate and the layers were separated. The aqueous layer wasextracted with ethyl acetate. The organic layers were combined, washedwith brine, dried over Na₂SO₄ and concentrated under reduced pressure.The crude mixture was purified by flash chromatography (silica gel,eluent: 1:4→1:1→3:1 EtOAc:Hexanes) to afford 17,18-unsaturatedphthalazine 53 (16.3 mg, 85%). ¹H NMR (500 MHz, CDCl₃) Shift=9.51 (d,J=5.0 Hz, 2H) 8.02 (d, J=5.0 Hz, 1H), 7.91 (s, 1H), 7.90 (d, J=5.0 Hz,1H), 6.37 (br. s., 1H), 5.80 (br. s., 1H), 5.37 (br. m., 1H), 3.98 (m,4H), 2.75 (m, 1H), 2.59-2.52 (m, 2H), 2.50-2.42 (m, 3H), 2.37-2.26 (m,3H), 2.14 (d, J=15.0 Hz, 1H), 2.00 (dd, J=15.0, 2.5 Hz, 1H), 1.93 (m,1H), 1.79 (m, 1H), 1.72-1.66 (m, 2H), 1.16 (s, 3H). HRMS (ESI) (m/z)calc'd for C₂₉H₃₁N₂O₃ [M+H]⁺: 455.5680. found 455.5679.

Synthesis of 17,18-Unsaturated Amidepyridine 47

To a solution of triflate 20 (20 mg, 42.1 μmol, 1.0 equiv) and3-aminopyridine (19.8 mg, 210 μmol, 5.0 equiv) in DMF (1 mL) was addedtriethylamine (12 μL, 84.3 μmol, 2.0 equiv) and Pd(dppf)Cl₂.CH₂Cl₂ (1.72mg, 2.10 μmol, 0.05 equiv). Reaction flask was installed with CO balloonand the solution was purged for 5 min at room temperature. Then, thereaction mixture was heated up to 85° C. and stirred for 4 h. Themixture was allowed to cool to room temperature and EtOAc (3 mL) and H₂Owas added. The layers were separated and the aqueous layer was extractedwith EtOAc (3×2 mL), and the combined organic layers were washed withbrine (3 mL), dried over Na₂SO₄, and concentrated under reducedpressure. The crude mixture was purified by flash column chromatography(silica gel, eluent: 10:10:1→10:10:2 Hexanes:EtOAc:MeOH) to provide17,18-unsaturated amidepyridine 47 (17 mg, 89%). ¹H NMR (500 MHz, CDCl₃)Shift=8.57 (d, J=2.4 Hz, 1H), 8.33 (dd, J=1.2, 4.6 Hz, 1H), 8.20 (d,J=8.3 Hz, 1H), 7.70 (s, 1H), 7.26 (dd, J=4.9, 7.8 Hz, 1H), 6.55 (br. s.,1H), 5.77 (s, 1H), 5.36 (d, J=2.9 Hz, 1H), 4.03-3.89 (m, 4H), 2.63-2.56(m, 2H), 2.52 (ddd, J=2.9, 7.3, 17.1 Hz, 1H), 2.52-2.37 (m, 3H),2.36-2.27 (m, 2H), 2.20 (t, J=12.2 Hz, 1H), 2.12 (d, J=13.2 Hz, 1H),1.97 (dd, J=2.2, 12.9 Hz, 1H), 1.89 (td, J=8.8, 13.2 Hz, 1H), 1.78 (tdd,J=2.4, 4.8, 12.8 Hz, 1H), 1.71-1.62 (m, 2H), 1.11 (s, 3H). HRMS (ESI)(m/z) calc'd for C₂₇H₃₁N₂O₄ [M+H]⁺: 447.2278. found 447.2289.

Example 4 General Method for Synthesis of Ketones

To a solution of ketal in THF at 0° C. was added 6N HCl (THF:6N HCl=1:1,0.05M). The mixture was warmed to room temperature and stirred for 1 h.The reaction was quenched with 6 N NaOH and ethyl acetate and the layerswere separated. The aqueous layer was extracted with ethyl acetate. Theorganic layers were combined dried over Na₂SO₄ and concentrated underreduced pressure.

Synthesis of Ketone 45

The crude mixture was purified by flash column chromatography (silicagel, eluent: 15:1 Dichloromethane:MeOH) to afford ketone 45 (8.5 mg,95%). ¹H NMR (500 MHz, CDCl₃) Shift=8.60 (s, 1H), 8.52 (d, J=3.9 Hz,1H), 7.74 (br. s., 1H), 7.28 (dd, J=4.4, 8.3 Hz, 1H), 5.91 (s, 1H), 5.37(dd, J=2.7, 4.6 Hz, 1H), 3.88 (d, J=13.7 Hz, 1H), 3.84 (d, J=13.7 Hz,1H), 2.91 (d, J=14.6 Hz, 1H), 2.82 (t, J=9.0 Hz, 1H), 2.65 (d, J=15.1Hz, 1H), 2.64 (dd, J=10.3, 14.6 Hz, 1H), 2.59-2.41 (m, 3H), 2.32-2.20(m, 3H), 2.19-2.07 (m, 3H), 1.85-1.72 (m, 2H), 1.72-1.61 (m, 2H), 1.44(br. s., 1H), 0.82 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₅H₃₁N₂O₂[M+H]⁺: 391.2380. found 391.2366.

Synthesis of Ketone 54

The crude mixture was purified by flash column chromatography (silicagel, eluent: 1:1 EtOAc:Hexanes) to afford ketone 54 (8.7 mg, 80%). ¹HNMR (500 MHz, CDCl₃) Shift=9.54 (d, J=10.0 Hz, 2H) 8.04 (d, J=10.0 Hz,1H), 7.93 (br. s., 2H), 6.40 (br. s., 1H), 5.95 (br. s., 1H), 5.47 (br.m., 1H), 2.95 (d, J=10.0 Hz, 1H), 2.77 (m, 1H), 2.71-2.62 (m, 2H),2.59-2.44 (m, 7H), 2.34 (t, J=10.0 Hz, 1H), 2.19 (t, J=10.0 Hz, 1H),2.00 (m, 1H), 1.78 (m, 1H), 1.18 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₇H₂₇N₂O₂ [M+H]⁺: 411.5155. found 411.5152.

Synthesis of Ketone 57

The crude mixture was purified by flash column chromatography (silicagel, eluent: 1:1 EtOAc:Hexanes) to afford ketone 57 (13.7 mg, 72%). ¹HNMR (500 MHz, CDCl₃) Shift=8.04 (br. s., 1H), 7.68 (d, J=10.0 Hz, 1H),7.48 (br. s., 1H), 7.27 (d, J=10.0 Hz, 1H), 6.13 (br. s., 1H), 5.93 (br.s., 1H), 5.45 (br. t., J=5 Hz, 1H), 2.97 (d, J=15.0 Hz, 1H), 2.76-2.64(m, 3H), 2.53-2.43 (m, 6H), 2.40-2.34 (m, 2H), 2.17 (t, J=10.0 Hz, 1H),1.98 (m, 1H), 1.77 (m, 1H), 1.11 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₆H₂₇N₂O₂ [M+H]⁺: 399.5048. found 399.5047.

Synthesis of Ketone 60

The crude mixture was purified by flash column chromatography (silicagel, eluent: 1:1→3:1 EtOAc:Hexanes, buffered with 2% triethylamine) toafford ketone 60 (3.3 mg, 68%). ¹H NMR (500 MHz, CDCl₃) Shift=9.93-10.27(br s, 1H), 8.06 (s, 1H), 7.75 (s, 1H), 7.47 (ABq, J_(AB)=8.8 Hz,Δν=29.5 Hz, 2H), 6.03 (dd, J=2.0, 2.0 Hz, 1H), 5.94 (s, 1H), 5.47 (dd,J=3.9, 3.9 Hz, 1H), 2.97 (d, J=15.1 Hz, 1H), 2.63-2.76 (m, 3H),2.53-2.59 (m, 1H), 2.44-2.50 (m, 5H), 2.35-2.42 (m, 2H), 2.17 (dd,J=9.3, 9.3 Hz, 1H), 1.95-2.01 (m, 1H), 1.74-1.80 (m, 1H), 1.11 (s, 3H).HRMS (ESI) (m/z) calc'd for C₂₆H₂₇N₂O₂ [M+H]⁺: 399.2073. found 399.2043.

Synthesis of Ketone 22

For the method, see ‘Synthesis of Ketone 13’. The resulting residue wasthen purified by flash chromatography (silica gel, eluent: 3:2→1:2Hexanes:EtOAc) to afford ketone 22 (8.2 mg, 61%). ¹H NMR (500 MHz,CDCl₃) Shift=9.24 (br. s., 1H), 8.51 (d, J=5.4 Hz, 1H), 7.94 (s, 1H),7.84-7.76 (m, 2H), 7.63 (d, J=5.4 Hz, 1H), 6.27 (br. s., 1H), 5.97 (s,1H), 5.50 (dd, J=2.4, 4.9 Hz, 1H), 2.98 (d, J=14.6 Hz, 1H), 2.78 (dd,J=6.8, 11.2 Hz, 1H), 2.71 (d, J=14.6 Hz, 1H), 2.72-2.63 (m, 1H), 2.61(d, J=5.4 Hz, 1H), 2.59-2.54 (m, 2H), 2.54-2.50 (m, 2H), 2.50-2.42 (m,2H), 2.39 (ddd, J=1.5, 11.0, 12.9 Hz, 1H), 2.20 (ddd, J=1.5, 9.5, 11.5Hz, 1H), 2.01 (ddd, J=7.3, 8.8, 12.7 Hz, 1H), 1.79 (dt, J=7.3, 11.2 Hz,1H), 1.18 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₈H₂₈NO₂ [M+H]⁺:410.2115. found 410.2111.

Synthesis of Ketone 48

For the method, see ‘Synthesis of Ketone 13’. The resulting residue wasthen purified by flash chromatography (silica gel, eluent: 20:10:3Hexanes:EtOAc:2M NH₃ solution in MeOH) to afford ketone 48 (5.0 mg,74%). ¹H NMR (500 MHz, CDCl₃) Shift=8.60 (d, J=2.0 Hz, 1H), 8.37 (dd,J=1.0, 4.9 Hz, 1H), 8.24-8.20 (m, 1H), 7.55 (s, 1H), 7.30 (dd, J=4.9,8.3 Hz, 1H), 6.57 (br. s., 1H), 5.94 (s, 1H), 5.48 (dd, J=2.2, 5.1 Hz,1H), 2.95 (d, J=15.1 Hz, 1H), 2.69 (d, J=14.6 Hz, 1H), 2.68 (d, J=12.7Hz, 1H), 2.66-2.61 (m, 2H), 2.61-2.53 (m, 2H), 2.52-2.44 (m, 3H), 2.41(d, J=18.1 Hz, 1H), 2.29 (ddd, J=1.5, 11.1, 12.8 Hz, 1H), 2.22-2.15 (m,J=1.5, 9.4, 11.1 Hz, 1H), 1.98 (ddd, J=7.6, 9.0, 12.7 Hz, 1H), 1.76 (dt,J=7.3, 11.2 Hz, 1H), 1.14 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₅H₂₇N₂O₃ [M+H]⁺:403.2016. found 403.2023.

Example 5 General Method for Synthesis of N-Oxides

To a solution of amine (1.00 equiv) in methanol (0.028 M) was added H₂O₂(32.0 equiv) at room temperature. After 25 h, saturated NaHCO₃ solutionwas added, diluted with dichloromethane, and the layers were separated.The aqueous layer was extracted with dichloromethane. The organic layerswere combined, washed with brine, dried over Na₂SO₄ and concentratedunder reduced pressure.

Synthesis of 14B—N-oxide (14BNO)

The crude mixture was purified by flash column chromatography (silicagel, eluent: 90:9:1→80:18:2 Chloroform:Methanol:5N NH₄OH solution inH₂O) to provide N-oxide 14BNO (23.5 mg, 95%). ¹H NMR (500 MHz, CDCl₃)Shift=9.21 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.77 (s, 1H), 7.75 (d, J=8.3Hz, 1H), 7.61 (d, J=5.9 Hz, 1H), 7.57 (dd, J=1.0, 8.8 Hz, 1H), 5.76 (s,1H), 5.28 (d, J=2.9 Hz, 1H), 3.44-3.36 (m, 1H), 3.22 (s, 3H), 3.12 (t,J=10.8 Hz, 1H), 3.10 (d, J=1.0 Hz, 3H), 2.47 (dd, J=8.8, 11.2 Hz, 1H),2.44-2.29 (m, 5H), 2.28-2.13 (m, 4H), 2.09 (ddd, J=1.5, 9.3, 11.2 Hz,1H), 2.06-1.97 (m, 2H), 1.94 (dd, J=5.1, 17.4 Hz, 1H), 1.85 (dq, J=5.4,12.2 Hz, 1H), 1.83-1.76 (m, 1H), 1.72 (td, J=9.3, 12.2 Hz, 1H), 0.54 (s,3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₇N₂O₂ [M+H]⁺:457.2850. found457.2842.

Synthesis of 14A-N-oxide (14ANO)

The crude mixture was purified by flash column chromatography (silicagel, eluent: 90:9:1→80:18:2 Chloroform:Methanol:5N NH₄OH solution inH₂O) to provide N-oxide 14ANO (3.6 mg, 77%). ¹H NMR (500 MHz, CDCl₃)Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz, 1H), 7.80 (s, 1H), 7.77 (d, J=8.8Hz, 1H), 7.64 (d, J=5.9 Hz, 1H), 7.60 (d, J=8.8 Hz, 1H), 5.81 (s, 1H),5.36 (d, J=2.9 Hz, 1H), 3.46 (t, J=12.4 Hz, 1H), 3.24 (s, 3H), 3.18 (s,3H), 3.16 (t, J=9.8 Hz, 1H), 2.64 (dd, J=3.2, 7.1 Hz, 1H), 2.59-2.47 (m,3H), 2.43-2.28 (m, 4H), 2.28-2.15 (m, 2H), 2.09-1.99 (m, 2H), 1.97 (dd,J=5.1, 12.4 Hz, 1H), 1.88 (dq, J=5.4, 12.2 Hz, 1H), 1.81-1.71 (m, 2H),1.51 (dq, J=4.1, 12.3 Hz, 1H), 0.56 (s, 3H). HRMS (ESI) (m/z) calc'd forC₃₀H₃₇N₂O₂ [M+H]⁺:457.2850. found 457.2846.

Cortistatin A N-Oxide Formation

Cortistatin A N-Oxide:

To a solution of cortistatin A (2 mg) in ethyl acetate (1 mL) was addedAldrich silica gel Davisil™ (200 mesh) (200 mg) and this solution wasstirred exposed to air for 1 hour. Silica gel was filtered and thefiltrate was concentrated to give crude cortistatin A N-oxide that wasfurther purified by SiO₂ chromatography (eluent: 50% methanol/ethylacetate) to afford cortistatin A N-oxide (1.8 mg, 90% yield). ¹H NMR(500 MHz, CDCl₃) δ 9.22 (s, 1H), 8.50 (d, J=5.8 Hz, 1H), 7.78 (s, 1H),7.76 (d, J=8.8 Hz, 1H), 7.63 (d, J=5.9 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H),6.28 (s, 1H), 5.49 (m, 1H), 4.15 (d, J=9.3 Hz, 1H), 3.83 (t, J=9.8, 9.8Hz, 1H), 3.31-3.36 (m, 1H), 3.26 (s, 3H), 3.19 (s, 3H), 3.16 (dd, J=9.3,9.3 Hz, 1H), 2.50 (dd, J=11.7, 8.8 Hz, 1H), 2.14-2.40 (m, 5H), 1.97-2.07(m, 3H), 1.81-1.90 (m, 2H), 1.68-1.75 (m, 1H), 1.49-1.65 (m, 1H), 0.55(s, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₇N₂O₄ [M+H]⁺: 489.2753. found489.5928.

Example 6 Synthesis of C3-Alcohols and Substituted Analogs

Synthesis of β-Alcohol 17B

β-Alcohol 17B:

A solution of ketone 13 (2.00 mg, 4.85 μmol, 1.0 equiv) in THF (350 μL)was cooled to −78° C. and a solution of L-selectride in THF (1 M, 9.71μL, 9.71 μmol, 2.0 equiv) was added. After 1 h, saturated NH₄Cl solution(400 μL) and ethyl acetate (300 μL) was added, which was allowed to warmto room temperature. The aqueous phase was extracted with ethyl acetate(3×1 mL) and the combined organic phases were washed with brine (1 mL),dried over Na₂SO₄, and concentrated under reduced pressure. The residuewas purified by preparative TLC (eluent: 1:1 Hexanes:EtOAc) to affordβ-alcohol 17B (ca. 1.2 mg, 60%).

¹H NMR (600 MHz, CDCl₃) Shift=9.26 (br. s, 1H), 8.49 (br. s, 1H), 7.82(s, 1H), 7.78 (d, J=8.2 Hz, 1H), 7.69 (br. s, 1H), 7.63 (d, J=7.6 Hz,1H), 5.75 (s, 1H), 5.26 (br. s, 1H), 4.36 (br. s, 1H), 3.15 (t, J=9.7Hz, 1H), 2.63 (t, J=13.5 Hz, 1H), 2.51 (dd, J=9.1, 10.9 Hz, 1H),2.42-2.28 (m, 2H), 2.24 (t, J=10.6 Hz, 1H), 2.21-2.12 (m, 2H), 2.12-1.97(m, 3H), 1.93 (dd, J=5.0, 17.3 Hz, 2H), 1.90-1.80 (m, 2H), 1.79-1.58 (m,3H), 0.54 (s, 2H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₂NO₂ [M+H]⁺:414.2428. found 414.2436.

α-Alcohol 17A

A solution of ketone 13 (9.6 mg, 23.3 μmol, 1.00 equiv) in THF (750 μL)was cooled to −78° C. and a solution of LAH in diethyl ether (1.0 M,35.0 μL, 35.0 μmol, 1.50 equiv) was added. After 10 min, saturated NH₄Clsolution (500 μL) and ethyl acetate (500 μL) was added, which wasallowed to warm to room temperature. The aqueous phase was extractedwith ethyl acetate (3×1 mL) and the combined organic phases were washedwith brine (1 mL), dried over Na₂SO₄, and concentrated under reducedpressure. The residue was purified by flash column chromatography(silica gel, eluent: 1:1→1:5 Hexanes:EtOAc→100% EtOAc) to provideα-alcohol 17A (8.5 mg, 88%). ¹H NMR (500 MHz, CDCl₃) Shift=9.22 (s, 1H),8.47 (d, J=5.3 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J=8.2 Hz, 1H), 7.63 (d,J=5.9 Hz, 1H), 7.59 (dd, J=1.2, 8.8 Hz, 1H), 5.75 (s, 1H), 5.28 (d,J=2.3 Hz, 1H), 3.78 (tt, J=4.0, 11.3 Hz, 1H), 3.14 (t, J=10.0 Hz, 1H),2.51 (dd, J=8.5, 11.4 Hz, 1H), 2.40-2.33 (m, 2H), 2.32 (dt, J=4.7, 12.3Hz, 1H), 2.28-1.98 (m, 7H), 1.93 (dd, J=5.0, 17.3 Hz, 1H), 1.90-1.81 (m,2H), 1.74-1.62 (m, 2H), 1.40 (dtd, J=5.9, 11.6, 13.8 Hz, 1H), 0.53 (s,3H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₂NO₂ [M+H]⁺: 414.2428. found414.2437.

α-Methylether 64A

To a solution of α-alcohol 17A (2.0 mg, 4.83 μmol, 1.00 equiv) in DMF(300 μL) was added 60 wt % NaH (1.0 mg, 24.1 μmol, 5.00 equiv) at roomtemperature and pre-stirred 30 min. Temperature was lowered to −10° C.and MeI (2.0 μL, 29.0 μmol, 6.00 equiv) was added. After 2.5 hours, 2 MNaOH solution (200 μL) and ethyl acetate (500 μL) was added, which wasallowed to warm to room temperature. The aqueous phase was extractedwith ethyl acetate (3×1 mL) and the combined organic phases were washedwith brine (1 mL), dried over Na₂SO₄, and concentrated under reducedpressure. The residue was purified by preparative TLC (eluent: 1:2Hexanes:EtOAc) to afford α-methylether 64A (ca. 1.2 mg, 58%). ¹H NMR(600 MHz, CDCl₃) Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79 (s,1H), 7.75 (d, J=8.8 Hz, 1H), 7.63 (d, J=5.9 Hz, 1H), 7.59 (dd, J=1.5,8.5 Hz, 1H), 5.74 (s, 1H), 5.28 (d, J=2.9 Hz, 1H), 3.39 (s, 3H), 3.29(tt, J=3.5, 11.4 Hz, 1H), 3.14 (t, J=10.0 Hz, 1H), 2.52 (dd, J=8.5, 11.4Hz, 1H), 2.42-2.29 (m, 3H), 2.25 (t, J=11.7 Hz, 1H), 2.24-2.08 (m, 5H),2.06 (td, J=4.1, 12.9 Hz, 1H), 1.93 (dd, J=5.3, 17.0 Hz, 1H), 1.86 (dq,J=5.3, 12.3 Hz, 1H), 1.79 (t, J=12.0 Hz, 1H), 1.75-1.61 (m, 2H), 1.32(dtd, J=4.7, 11.5, 14.0 Hz, 1H), 0.53 (s, 3H). HRMS (ESI) (m/z) calc'dfor C₂₈H₃₂NO₂ [M+H]⁺: 428.2584. found 428.2573.

β-Methylether 64B

The reaction condition is same as the synthesis of α-methylether 64A.The residue was purified by preparative TLC (eluent: 1:2 Hexanes:EtOAc)to afford C3 β-methylether 64B (1.2 mg, 58%). ¹H NMR (500 MHz, CDCl₃)Shift=9.22 (s, 1H), 8.48 (d, J=5.9 Hz, 1H), 7.79 (s, 1H), 7.76 (d, J=8.8Hz, 1H), 7.63 (d, J=5.9 Hz, 1H), 7.60 (dd, J=1.5, 8.3 Hz, 1H), 5.74-5.70(m, 1H), 5.25 (dd, J=2.2, 5.1 Hz, 1H), 3.75-3.69 (m, 1H), 3.35 (s, 3H),3.18-3.10 (m, J=9.3 Hz, 1H), 2.51 (dd, J=8.5, 11.5 Hz, 1H), 2.52-2.44(m, 1H), 2.40-2.26 (m, 4H), 2.17 (s, 3H), 2.14-2.08 (m, 1H), 2.07-1.96(m, 2H), 1.96-1.90 (m, 2H), 1.86 (dq, J=4.9, 12.0 Hz, 1H), 1.76-1.61 (m,1H), 1.55-1.45 (m, 1H), 0.54 (s, 3H). HRMS (ESI) (m/z) calc'd forC₂₈H₃₂NO₂ [M+H]⁺: 428.2584. found 428.2599.

α-Monomethylcarbamate 68A

To a solution of α-alcohol 17A (3.5 mg, 8.5 μmol, 1.00 equiv) in CH₃CN(350 μL) was added CDI (2.1 mg, 12.7 μmol, 1.50 equiv) and the reactionmixture was refluxed for 4 hours. The crude mixture was concentratedunder reduced pressure and used for the next reaction without furtherpurification.

To a solution of the crude mixture in DCM (300 μL) was added MeNH₂ inTHF (2 M, 50 μL) at room temperature and stirred 14 hours. The crudemixture was concentrated under reduced pressure and purified bypreparative TLC (eluent: 1:1 Hexanes:EtOAc) to afford C3α-monomethylcarbamate 68A (2.4 mg, 60% in 2 steps). ¹H NMR (500 MHz,CDCl₃) Shift=9.28 (s, 1H), 8.50 (d, J=5.9 Hz, 1H), 7.87 (s, 1H), 7.83(d, J=8.8 Hz, 1H), 7.75 (d, J=5.9 Hz, 1H), 7.69 (d, J=8.8 Hz, 1H), 5.77(s, 1H), 5.30 (dd, J=2.2, 4.6 Hz, 1H), 4.76 (t, J=11.7 Hz, 1H), 4.61(br. s., 1H), 3.17 (t, J=10.0 Hz, 1H), 2.82 (d, J=4.4 Hz, 3H), 2.53 (dd,J=8.5, 11.5 Hz, 1H), 2.38 (d, J=17.1 Hz, 2H), 2.32 (dd, J=3.7, 11.5 Hz,1H), 2.30-2.14 (m, 5H), 2.13-2.01 (m, 2H), 1.95 (dd, J=5.1, 17.3 Hz,1H), 1.94-1.82 (m, 2H), 1.78-1.67 (m, 2H), 1.43 (dq, J=4.9, 12.7 Hz,1H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'd for C₃₀H₃₅N₂O₃ [M+H]⁺:471.2642. found 471.2631.

α-Methoxyethylether 63A

To a solution of α-alcohol 17A (2.0 mg, 4.83 μmol, 1.00 equiv) in DMF(300 μL) was added 60 wt % NaH (1.0 mg, 24.1 μmol, 5.00 equiv) at roomtemperature and pre-stirred 30 min before the addition of MeO(CH₂)₂Br(1.6 μL, 16.6 μmol, 3.00 equiv). After 36 hours, 2 M NaOH solution (200μL) and ethyl acetate (500 μL) was added. The aqueous phase wasextracted with ethyl acetate (3×1 mL) and the combined organic phaseswere washed with brine (1 mL), dried over Na₂SO₄, and concentrated underreduced pressure. The residue was purified by preparative TLC (eluent:2:5 Hexanes:EtOAc) to afford C3 α-methoxyethylether 63A (ca. 0.7 mg,27%). ¹H NMR (600 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.9 Hz,1H), 7.81 (s, 1H), 7.77 (d, J=8.3 Hz, 1H), 7.65 (d, J=5.9 Hz, 1H), 7.61(d, J=8.8 Hz, 1H), 5.75 (s, 1H), 5.29 (d, J=2.4 Hz, 1H), 3.74-3.63 (m,2H), 3.61-3.51 (m, 2H), 3.44 (tt, J=3.9, 11.7 Hz, 1H), 3.44-3.39 (s,3H), 3.16 (t, J=9.8 Hz, 1H), 2.54 (dd, J=8.5, 11.5 Hz, 1H), 2.43-2.30(m, 3H), 2.30-2.17 (m, 4H), 2.17-2.02 (m, 3H), 1.95 (dd, J=5.1, 17.3 Hz,1H), 1.92-1.82 (m, 2H), 1.78-1.62 (m, J=8.3 Hz, 2H), 1.41 (dtd, J=4.1,11.9, 13.5 Hz, 1H), 0.55 (s, 3H). HRMS (ESI) (m/z) calc'd for C₂₈H₃₂NO₂[M+H]⁺: 472.2846. found 472.2850.

Example 7 Synthesis of Amines from Alcohols α-Dimethylhydantoin 74A

To a solution of β-alcohol 17B (5.0 mg, 12.3 μmol, 1.0 equiv) in THF(400 μL) was added dimethylhydantoin (7.8 mg, 61.6 μmol, 5.0 equiv) andPPh₃ (9.7 mg, 36.9 μmol, 3.0 equiv). Reaction mixture was cooled to 0°C. and DEAD (16.1 μL of 40 wt % solution in toluene, 36.9 μmol, 3.0equiv). Reaction was warmed up to 50° C. and stirred 17 h. After coolingthe reaction mixture to room temperature, 1N NaOH solution (300 μL) andwas added and the aqueous phase was extracted with ethyl acetate (3×0.5mL) and the combined organic phases were washed with brine (1 mL), driedover Na₂SO₄, and concentrated under reduced pressure. The crude mixturewas purified by preparative TLC (silica gel, eluent: 40:1MeOH:Dichloromethane) to afford α-dimethylhydantoin 74A (0.9 mg, 14%).¹H NMR (500 MHz, CDCl₃) Shift=9.24 (s, 1H), 8.50 (d, J=5.4 Hz, 1H), 7.81(s, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.64 (d, J=5.4 Hz, 1H), 7.61 (dd,J=1.5, 8.8 Hz, 1H), 5.79 (d, J=1.5 Hz, 1H), 5.32 (dd, J=2.7, 5.1 Hz,1H), 5.18 (s, 1H), 4.10 (tdd, J=3.2, 11.3, 13.1 Hz, 1H), 3.16 (dd,J=9.3, 10.7 Hz, 1H), 2.86 (t, J=12.7 Hz, 1H), 2.54 (dd, J=8.3, 11.7 Hz,1H), 2.45 (dd, J=2.9, 14.6 Hz, 1H), 2.30 (br. s., 6H), 2.19 (tq, J=4.4,9.0 Hz, 1H), 2.09-2.00 (m, 1H), 1.96 (dd, J=5.4, 17.6 Hz, 1H), 1.92-1.80(m, 2H), 1.80-1.64 (m, 3H), 1.42 (d, J=4.9 Hz, 6H), 0.56 (s, 3H). HRMS(ESI) (m/z) calc'd for C₃₃H₃₈N₃O₃ [M+H]⁺: 524.2908. found 524.2892.

Biological Methods Cell Culture and Cell Lines

MV4;11, RS4;11, HL-60, K562, L-Wnt3A and L cells were obtained fromATCC. HEL, NB4, KG1, UKE-1, UKE-1per, SET-2 and SET-2per cells wereprovided by Ross Levine (MSKCC). MOLM-14 was provided by Scott Armstrong(Boston Children's Hospital). MV4;11-mCLP cells were provided by AndrewKung (Dana Farber Cancer Institute). HEK293 STF cells were provided byJeremy Nathans (Johns Hopkins University School of Medicine). All cellswere maintained in a humidified incubator set to 37° C., 5% CO₂. MV4;11,RS4;11, K562, HEL, NB4, MOLM-14 and KG1 cells were grown in RPMI-1640with 10% FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. HaCaTand HEK293 STF cells were grown in DMEM with 10% FBS, 100 U ml⁻¹penicillin, and 100 μg ml⁻¹ streptomycin, with 200 μg ml⁻¹ G418 forHEK293STF cells. L-Wnt3A and L cells were grown in DMEM with 10% FBS,100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 400 μg ml⁻¹ G418(for L-Wnt3A only). SET-2 cells were grown in RPMI-1640 with 20% FBS,100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. HL-60 were grown inIMDM with 20% FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin.UKE-1 cells were grown in RPMI-1640 with 10% FBS, 10% horse serum, 100 Uml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 1 μM hydrocortisone.SET-2per and UKE-1per cells were maintained in the presence of 0.7 μMand 1 μM ruxolitinib, respectively. Human Umbilical Vein EndothelialCells (HUVECs) were obtained from Life Technologies and cultured in M200medium with low serum growth supplement (LSGS, Life Technologies).

Reagents

Compounds for testing were prepared as 100% DMSO stock solutions andstored under argon at −80° C. Doxorubicin HCl, anti-FLAG (F1804) andanti-Actin (A5060) antibodies were obtained from Sigma-Aldrich.Anti-Smad 2/3 (8565), anti-Stat1 (9172), Anti-phospho-Stat1 Tyr701(9171), and anti-phospho-Stat1 Ser727 (9177) antibodies were obtainedfrom Cell Signaling Technologies. Anti-phospho-Smad 2/3 T220/T179 wasobtained from Rockland, IFNγ was obtained from Life Technologies andTGFβ1 was obtained from R&D Systems. Wnt3A conditioned media and L-cellconditioned media were obtained as described by ATCC.

Plasmids, Mutagenesis, Packaging and Transduction.

5′-FLAG-tagged CDK8 was PCR amplified from pBabe.puro.CDK8.flag(Addgene, original source Firestein et al., Nature (2008) 455, 547-551)with the addition of EcoRI (5′) and XbaI (3′) restriction sites andcloned into the lentiviral expression vector pLVX-EF1alpha-IRES-mCherry(Clonetech) by sticky-end ligation (EcoRI and XbaI) and transformed intoE. coli (One Shot Stbl3, Invitrogen). 5′-FLAG-tagged CDK19 was PCRamplified from F-CDK8L (Addgene, original source Conaway et al., FEBSLetters (2005) 579, 904-908) with the addition of SpeI (5′) and XbaI(3′) restriction sites and cloned into the lentiviral expression vectorpLVX-EF1alpha-IRES-ZsGreen1 (Clonetech) by sticky-end ligation andtransformed into E. coli (One Shot Stbl3, Invitrogen). Point mutationswere introduced by whole plasmid PCR (QuikChange II XL Site-DirectedMutagenesis Kit, Agilent). 5′-FLAG-tagged CDK8 or CDK19 mutant wassubsequently PCR amplified, using EF1alpha forward and IRES reverseprimers (see Table 1), and cloned into pLVX-EF1 alpha-IRES-mCherry orpLVX-EF1alpha-IRES-ZsGreen1, respectively, and transformed into E. coli(One Shot Stbl3, Invitrogen). Primers used to clone CDK8 and CDK19 andintroduce the W105M point mutation into each protein are provided inTable 1.

TABLE 1 Primers used to clone CDK8 and CDK19 and introduce W105Mpoint mutation Primer Sequence 1^(st) round 5′ EcoRIGAATTCGCCACCATGGACTA CDK8 (SEQ ID NO.: 3): 1^(st) round 3′ XbaITCTAGATCAGTACCGATGTGTCT CDK8 (SEQ ID NO.: 4): 2^(nd) round 5′ EcoRITAGCTAGAATTCGCCACCATG CDK8 (SEQ ID NO.: 5): 2^(nd) round 3′ XbaIGTCGAGTCTAGATCAGTACCG CDK8 (SEQ ID NO.: 6): 1^(st) round 5′ SpeIACTAGTATGCCAGACTACAAGGA CDK19 (SEQ ID NO.: 7): 1^(st) round 3′ XbaITCTAGATCAGTACCGGTGGG CDK19 (SEQ ID NO.: 8): 2^(nd) round 5′ SpeIGTCGAGACTAGTATGCCAGAC CDK19 (SEQ ID NO.: 9): 2^(nd) round 3′ XbaITCGAGTCTAGATCAGTACCGG CDK19 (SEQ ID NO.: 10): EF1alpha forwardTCAAGCCTCAGACAGTGGTTC (SEQ ID NO.: 11): IRES reverse (SEQACGTGTATAAGATACACCT ID NO.: 12): CDK8 W105M:CTATGCTGAACATGACCTCATGCATATAATCAAGTTTCAC Trp105 (TGG)- >Met (ATG)(forward) (SEQ ID NO.: 13): CDK8 W105M:GTGAAACTTGATTATATGCATGAGGTCATGTTCAGCATAG Trp105 (TGG)- >Met (ATG)(reverse) (SEQ ID NO.: 14): CDK19 W105M:GCAGAGCATGACTTGATGCATATTATTAAGTTTCACC Trp105 (TGG)- >Met (ATG)(forward) (SEQ ID NO.: 15): CDK19 W105M:GGTGAAACTTAATAATATGCATCAAGTCATGCTCTGC Trp105 (TGG)- >Met (ATG)(reverse) (SEQ ID NO.: 15):

The lentiviral expression vectors were packaged by the University ofMassachusetts RNAi Core Facility by cotransfection of 293T cells withpackaging vector psPAX2 (Addgene) and envelope vector pMD2.G (Addgene).48 h after transfection, viral supernatants were collected, passedthrough 0.45 μm filter (Millipore) and used for transduction (MOI 2 for293T cells). Transductions were performed with RetroNectin (Clontech),according to the manufacturers instructions. Briefly, sterile 24-wellplates were coated with 500 μL of 20 μg/ml RetroNectin in sterile PBSand stored at 4° C. overnight. The next day, the RetroNectin solutionwas aspirated and the plates were blocked with 2% BSA in sterile PBS for30 min at room temperature. The plates were then washed 1× with PBS and300 to 500 μL of viral supernatant was added. The plates were thencentrifuged at 2000×g for 1.5 hr with a 1800 change in orientation after45 min. The plates were then incubated for 2 h in a humidified incubatorset to 37° C., 5% CO₂. The viral supernatant was then removed and 500 μLof 200,000 cells/ml was added to each well. After incubation of theplates for 1-3 days, the cells were expanded and the fluorescentprotein-expressing cells were isolated by FACS.

In order to determine whether CDK8 and CDK19 mediate theantiproliferative effects of cortistatin A or cortistatin A analogs,alleles of both mediator kinases were identified that would render cellsresistant to growth inhibition by cortistatin A. With guidance from ourcrystal structure, CDK8 Trp105 was selected for mutation to Met sincethe molecular modelling suggested that it would disrupt CDK8 binding tocortistatin A but not ATP. It was found that MOLM-14 cellsoverexpressing either FLAG-CDK8 W105M or FLAG-CDK19 W105M were highlydesensitized to growth inhibition of cortistatin A (>50-fold, FIG. 10A)and 14BNO (>50-fold, FIG. 22). Furthermore, CDK8 W105M was catalyticallycompetent yet far less sensitive to kinase inhibition by cortistatin Ain vitro (FIG. 10B) and in cells (FIG. 10C), thus proving that thesemutations act by reducing affinity for cortistatin A, but not ATP (FIG.10B). Collectively, these results provided unequivocal proof that CDK8and CDK19 act redundantly to mediate the antiproliferative effects ofcortistatin A. For FIGS. 10A and 22, cells were passaged and freshcompound was added on day 3 (mean+/−standard error, n=3). For FIG. 10B,FLAG-CDK8 and FLAG-CDK8 W105M were isolated from MOLM-14 cellsoverexpressing each protein and subjected to kinase reactions inpresence of the indicated concentrations of cortistatin A. For FIG. 10C,MOLM-14 cells expressing equal levels of FLAG-CDK8 or FLAG-CDK8 W105Mwere treated with the indicated concentrations of cortistatin A for 1hour followed by IFN-gamma for 1 hr. Cells were then collected, lysed,and analyzed by western blot.

Cell Growth Assays

HUVECs were plated in triplicate in 96-well plates at a density of 1,000cells/well in 75 μL. After 24 h, compounds were serially diluted inmedium and delivered to cells as 4× solutions in 25 μL (0.1% DMSO finalfor assay). 96 h after compound addition, CellTiter-Blue (Promega) wasadded to the wells, plates were incubated at 37° C. for 90 min, andfluorescence (555 nm excitation; 580 nm emission) was detected using aSPECTRAmax M3. IC₅₀ was determined by fitting the data to afour-parameter dose-response curve using GraphPad Prism 5.0.

For all other cell lines, compounds were serially diluted in medium anddispensed in triplicate in 96-well plates (0.1% DMSO final for assay)followed by the addition of exponentially growing cells at densitiesranging from 5,000 to 30,000 cells/well. Cells were also seeded into 2additional control wells containing vehicle (0.1% DMSO). After 3 days,the number of viable cells in one of the control wells was counted byhemocytometer or MOXI Z (Orflo) and an equal volume of cells for allwells were split-back with fresh vehicle/compounds such that theresulting seeding density for the control well was 5,000-30,000cells/well, matching the initial seeding density in that well. Thisprocedure was repeated on day 7 and day 10. % growth was calculated as %growth=100×(total estimated cell number in well−day 0 starting cellnumber)/(total average vehicle estimated cell number−day 0 starting cellnumber). To determine the estimated cell number in each well, cells thatwere counted in the control well were diluted 3-fold for an 8-pointdilution series, and transferred to 384-well plates in duplicate, 20 μLper well. Cells in all other wells were diluted 4-fold and transferredto 384-well plates in duplicate, 20 μL per well. 20 μL of CellTiter-Glo(Promega) was subsequently added to all wells and luminescence wasdetected using a SPECTRAmax M3. The control well dilution series was fitto a linear regression and used as a standard for calculating theestimated cell number for each well. For days 7 and 10, total estimatedcell number represents the split-adjusted theoretical cell number.

In Vitro CDK8 Kinase Assay

The in vitro kinase assay was performed as previously described (See,e.g., Knuesel et al., Mol. Cell. Biol. (2009) 29, 650-661). FLAG-CDK8and FLAG-CDK8 W105M were overexpressed and purified from MOLM-14 cells.

X-Ray Crystal Structure

The X-ray crystal structure of cortistatin A (CA) bound to CDK8/Cyclin Cwas obtained by Proteros Biostructures GmbH. Expression and purificationof CDK8/Cyclin C was performed as previously described (See, e.g.,Schneider et al., J. Mol. Biol. (2011) 412, 251-266). Diffraction datawere collected at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland).The structure was solved and refined to a final resolution of 2.40 Å.Data were processed using the programs XDS and XSCALE. The phaseinformation necessary to determine and analyze the structure wasobtained by molecular replacement with a previously solved structure ofCDK8/CycC as a search model. Subsequent model building and refinementwas performed with the software packages CCP4 and COOT. For thecalculation of the free R-factor, a measure to crossvalidate thecorrectness of the final model, about 3.4% of measured reflections wereexcluded from the refinement procedure. TLS refinement (using REFMAC5,CCP4) has been carried out, which resulted in lower R-factors and higherquality of the electron density map. The ligand parameterization andgeneration of the corresponding library files were carried out withCORINA. The water model was built with the “Find waters”-algorithm ofCOOT by putting water molecules in peaks of the F_(o)-F_(c) mapcontoured at 3.0 followed by refinement with REFMAC5 and checking allwaters with the validation tool of COOT. The criteria for the list ofsuspicious waters were: B-factor greater 80 Å², 2F_(o)-F_(c) map lessthan 1.2 σ, distance to closest contact less than 2.3 Å or more than 3.5Å. The suspicious water molecules and those in the active site (distanceto inhibitor less than 10 Å) were checked manually. The occupancy ofside chains, which were in negative peaks in the F_(o)-F_(c) map(contoured at −3.0 σ), were set to zero and subsequently to 0.5 if apositive peak occurred after the next refinement cycle. The RamachandranPlot of the final model shows 93.2% of all residues in the most favouredregion, 6.6% in the additionally allowed region, and 0.2% in thegenerously allowed region. No residues are found in the disallowedregion. Data collection, processing and refinement tables follow. Valuesin parenthesis refer to the highest resolution bin.

TABLE 2 Data collection and processing statistics for Cortistatin ALigand Cortistatin A X-ray source PXI/XO6SA (Swiss Light Source,Villigen, Switzerland) Wavelength [Å] 1.00004 Detector PILATUS 6MTemperature [K] 100      Space group P 2₁ 2₁ 2₁ Cell: a; b; c; [Å]70.49; 71.25; 171.25 α; β; γ; [°] 90.0; 90.0; 90.0 Resolution [Å] 2.40(2.65-2.40) Unique reflections 32875 (8656) Multiplicity 2.8 (2.8)Completeness [%] 94.9 (98.6) Rsym [%] 7.4 (44.8) Rmeas [%] 9.0 (54.8)Mean (I)/sd, calculated from 10.99 (2.66) independent reflections

TABLE 3 Refinement statistics for Cortistatin A Cortistatin A LigandResolution [Å] 85.62-2.40 Number of reflections (working/test)31676/1115 R_(crys) [%] 21.7 R_(free) [%], test-set contains 3.4% ofmeasured 26.6 reflections Total number of atoms: Protein 5017 Water 104Ligand 35 Formic acid 15 Deviation from ideal geometry (root mean squaredeviations from geometric target values): Bond lengths [Å] 0.009 Bondangles [°] 1.13 Bonded B's [Å], calculated with MOLEMAN 2.1 Ramachandranplot (calculated with PROCHECK): Most favoured regions [%] 93.2Additional allowed regions [%] 6.6 Generously allowed regions [%] 0.2Disallowed regions [%] 0.0

To understand how cortistatin A inhibits CDK8, we solved an X-raycrystal structure of the cortistatin A/CDK8/cyclin C ternary complex at2.40 Å resolution (FIG. 16A). Cortistatin A binds in the ATP-bindingsite of CDK8 and forms a single H-bond with CDK8 (dashed line, FIG. 16C)between the isoquinoline nitrogen of cortistatin A and the main chainamide N-H of Ala100 located in the hinge region. The remaining contactsbetween cortistatin A and CDK8 are hydrophobic, with cortistatin Aexhibiting remarkable shape complementarity with the ATP-binding site(FIG. 16B). In particular, the C13 angular methyl group and C5-C9 ethanebridge of cortistatin A perfectly fill hydrophobic cavities formed bythe side-chains of His106, Ala155, Trp105, and Asp103 (FIG. 16C). OtherCDKs do not have similar hydrophobic cavities to accommodate theC13-methyl and C5-C9 ethane bridge of cortistatin A, perhaps explainingthe impressive selectivity of cortistatin A for CDK8/CDK19.

There is also an apparent cation-□ interaction between the chargedC3-N,N-dimethylammonium ion of cortistatin A and Trp105 of CDK8, whichare within close contact (3.4 Å). CDK8 and CDK19 are the only CDKs withTrp at amino acid 105, suggesting that the cation-□ interaction as wellas hydrophobic contacts between cortistatin A and Trp105 might beimportant for the high affinity and selectivity of cortistatin A forCDK8. A closer view of the contact between Trp105 and theN,N-dimethylammonium ion of cortistatin A is shown in FIG. 16D. There isa direct contact between one methyl group of the N,N-dimethylamine ofcortistatin A and Trp105, which is perfect for a cation-□ interaction.

It is very surprising that 14B was as potent or more potent(approximately 2-fold more potent) than cortistatin A in cell growthinhibition and in CDK8 kinase inhibition since the C3 amine is now axialand might have been expected to clash with Trp105.

Western Blotting

For STAT1 studies, MOLM-14 cells were treated with vehicle (0.1% DMSO)or compound for 1 h followed by the addition of IFNγ (Life Technologies,PHC4031) for 1 h. Cells were then collected, washed twice with cold PBSand snap frozen in liquid nitrogen. For all Western blotting, whole-celllysates were prepared in RIPA buffer (Sigma-Aldrich, R0278) withphosphatase and protease inhibitor additives (Sigma-Aldrich, P8340,P0044, P5762) and protein concentration was determined by BCA assay(Thermo Scientific, 23225). RIPA extracts were resolved by denaturingpolyacrylamide gel electrophoresis (Life Technologies, NuPAGE) andtransferred to PVDF membrane (Millipore) using the Mini Trans-Blotsystem (BioRad). The membranes were probed as recommended by theantibody manufacturers and proteins detected by chemiluminescence usinghorseradish peroxidase-conjugated secondary antibodies. For SMAD2/3studies, HaCaT cells were treated as described for STAT1 with theaddition of TGFβ1 (R&D Systems) in place of IFNγ.

Reporter Assay

Exponentially growing HEK293 STF were plated in triplicate in 96-wellplates at a density of 5,000 cells/well in 75 μL. For experiments withazakenpaullone, after 24 h, cortistatin A was serially diluted in mediumcontaining 40 μM azakenpaullone and delivered to cells as 4× solutionsin 25 μL (0.2% DMSO and 10 μM azakenpaullone final for assay).Additional triplicate control wells on each plate had media withoutcells or cells without azakenpaullone. For experiments with Wnt3Aconditioned media, after 24 h, compounds were serially diluted in Wnt3Aconditioned media as 1× solutions (0.1% DMSO final). Old media wasremoved from the cells and replaced with the compound/Wnt3A-media.Additional triplicate control wells on each plate had media withoutcells or cells that were treated with L-conditioned media (no Wnt3A).After 24 h, the CellTiter-Fluor and Steady-Glo assays (Promega) weresubsequently performed in series to determine viability andluminescence, respectively, in each well (measurements made on aSPECTRAmax M3, Molecular Devices). Graphs were made in Prism 5 (GraphPadSoftware, Inc.) with nonlinear regression fit to a sigmoidaldose-response curve (variable slope).

Gene Expression

Exponentially growing MOLM-14, MV4;11, and K562 cells were seeded in12-well plates at a density of 500,000 to 800,000 cells/mL in 1 mL ofmedia followed by the addition of vehicle (0.1% DMSO) or cortistatin Aat 10 nM (“CA10”) for MOLM-14 and MV4;11 cells and 25 nM for K562 cellsin triplicate wells. After 24 h, cells were washed 2× with cold PBS,snap frozen, and RNA was isolated by RNeasy Plus Microkit (Qiagen) orTRIzol (Life Technologies). RNA was processed by the Dana Farber CancerInstitute Microarray Core Facility and hybridized to the Human U133 Plus2.0 microarray (Affymetrix). Microarrays were quality controlled usingthe affyQCReport Bioconductor package (See, e.g., Gentleman et al.,Genome Biol (2004) 5: R80; R Development Core Team, R: A Language andEnvironment for Statistical Computing. Vienna, Austria. ISBN3-900051-07-0). Satisfactory arrays were corrected for background,normalized, and log 2-transformed using the rma function of the affyBioconductor package (See, e.g., Rafael et al., Nucleic Acids Research(2003) 31:e15; Bolstad et al., Bioinformatics (2003) 19:185-193;Irizarry et al., Biostatistics (2003) 4: 249-264). Present/Absent callswere made using the mas5calls function of the affy package. Probe setspresent in >20% of samples and for which the interquartile rangewas >log 2(1.2) were retained for further analysis. Batch-correction bycell type (MOLM-14 vs. MV4;11) was performed with ComBat (See, e.g.,Johnson et al., Biostatistics (2007) 8:118-127). We used GenePattern forunsupervised hierarchical clustering of the samples based on Euclideandistance. Probe sets that were significantly up- or down-regulated atleast 1.5-fold in CA10-treated vs. DMSO control samples were identifiedusing the limma Bioconductor package (See, e.g., Smyth, G. K. (2005).Limma: linear models for microarray data. In: ‘Bioinformatics andComputational Biology Solutions using R and Bioconductor’. R. Gentleman,V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds), Springer, New York,2005). We set a p-value of <0.05, corrected for multiple hypothesistesting by the Benjamini-Hochberg method (See, e.g., Benjamini, Y. &Hochberg, Y. J. R. Stat. Soc., B (1995) 57:289-300) as our significancecut-off. Gene Set Enrichment Analysis (See, e.g., Subramanian et al.,Proc Natl Acad Sci USA. (2005) 102:15545-50) was carried out usingT-tests on log 2 values as the metric. Signatures included curated genesets (C2) downloaded from the Broad's MSigDB as well as signaturescurated in-house from literature.

In Vivo Studies

For pharmacokinetic studies with cortistatin A (CA), blood samples fromCD-1 mice were collected at the indicated times following i.p.administration of cortistatin A at 10 mg kg⁻¹ (FIG. 11A) and 1 mg/kg(FIG. 11B) and analyzed by HPLC/MS/MS to evaluate the concentration ofcortistatin A.

For pharmacokinetic studies with (14A), (14A) was formulated in 20%hydroxypropyl-beta-cyclodextrin (HPCD) and male CD-1 mice were given asingle dose of 14A at 3 mg/kg i.p. and intravenous (i.v.) and 10 mg/kgoral (p.o.). Prior to dosing, mice were fasted overnight until 4 hourspost-dosing. Blood samples were collected at the indicated timepointsand analyzed by HPLC/MS/MS to evaluate the concentration of (14A). SeeFIG. 24. From these studies, the following observations were made: (1)compound (14A) has an IV clearance in male CD-1 mice of 29 mL/min/kg,which is only ⅓^(rd) of hepatic blood flow in this species and indicatesthat (14A) has good stability; (2) the Vss is large (approximately 11.7L/kg) so (14A) has good tissue distribution; (3) the oralbioavailability was found to be approximately 44%; (4) the Cmax afteroral dosing was 255 ng/mL; (5) the t1/2 after oral dosing wasapproximately 5 hours (oral C24 trough concentrations were 14.2 ng/mL);and (5) IP bioavailability was 95%.

Efficacy studies using the MV4;11 xenograft model were performed aspreviously described. See, e.g., Etchin et al., Leukemia (2013) 27,66-74. Briefly, 2 million MV4;11 mCLP cells were introduced into7-week-old female NOD-SCID-IL2Rcγ^(null) (NSG) mice (The JacksonLaboratory) via tail vein injection. The tumor burden was assessed bybioluminescence imaging (BLI) using an IVIS Spectrum system (CaliperLife Sciences). 7 days-post injection, leukemia establishment wasdocumented by BLI and the mice were split into treatment groups toreceive either vehicle (20% hydroxypropyl-β-cyclodextrin) or cortistatinA (0.05 and 0.16 mg kg⁻¹) and treated once daily for 15 days. At 30 dayspost-treatment start date, 3 mice per group were sacrificed and bloodcounts were obtained using Hemavet 950 F instrument (Drew Scientific)and spleen, femur, and peripheral blood cells were collected andanalyzed by flow cytometry (LSR Fortessa, BD Biosciences). The mice anda portion of the spleen were also preserved in bouins forhistopathology. Mice were sectioned, paraffin-embedded and stained withhematoxylin and eosin. Survival of the drug-treated mice was measured asthe time from initiation of therapy until moribund state. Survivalbenefit was assessed by Kaplan-Meier analysis. For safety testing withcortistatin A (FIG. 13), female CD-1 mice were treated with eithervehicle (20% hydroxypropyl-β-cyclodextrin) or cortistatin A (0.16 mgkg⁻¹) once daily for 15 days and weighed daily. 2 h after the last dose,the mice were sacrificed, blood counts were obtained, blood chemistrywas analyzed and the mice were fixed with bouins for histopathology.Efficacy studies using the murine model of primary myelofibrosis(MPLW515L mice) were performed as previously described (See, e.g.,Marubayashi, et al., J. Clin, Invest. (2010) 120, 3578-3593) with oncedaily dosing with vehicle (20% hydroxypropyl-β-cyclodextrin) orcortistatin A. For the efficacy studies using the SET-2 subcutaneousxenograft model, 1×10⁷ SET-2 cells tumor cells in 50% matrigel wereinjected subcutaneously into the the flank of 8 to 12 week old femaleSCID Beige mice. Once tumors reached an average size of 80-120 mm³, themice were treated with vehicle or 0.16 mg kg⁻¹ daily for the duration ofthe study.

Liver Microsome Metabolic Stability Assay

Test compounds were incubated at 37° C. with liver microsomes pooledfrom multiple donors at 1 micromolar in the presence of a NADPHregenerating system at 0.5 mg/ml microsomal protein. At 0, 5, 10, 20,30, and 60 minutes, samples were removed, and mixed with coldacetonitrile containing an internal standard. After protein separation,samples were analyzed by LC/MS/MS for disappearance of the test compoundusing the ratio of peak area of analyte to internal standard. From thevalues, the microsome clearance was calculated. Species used were human,Sprague Dawley rat, beagle dog, and CD-1 mouse.

Human Hepatocyte Stability Assay

Test compounds were incubated in duplicate with cryopreservedhepatocytes at 37° C. The cells were thawed; viable cells counted, andequilibrated according to the supplier's directions. After 30 minequilibration at 37° C. with gentle agitation, the test compounds wereadded into the cells to give the desired final concentration of 1 μM.The cell suspension was incubated at 37° C. as above. At the indicatedtimes, samples were removed and mixed with an equal volume of ice-coldStop Solution (acetonitrile containing internal standards). Stoppedreactions were incubated at least ten minutes on ice, and an additionalvolume of water was added. The samples were centrifuged to removeprecipitated protein, and the supernatants were analyzed by LC/MS/MS toquantitate the remaining parent. Data were converted to % remaining bydividing by the time zero concentration value. Data were fit to afirst-order decay model to determine half-life. From a plot of log (In)peak area against time, the slope of the line was determined.Subsequently, half-life and intrinsic clearance were calculated usingthe equations below: Elimination rate constant (k)=(−slope); Half-life(T½) min=0.693/k; Intrinsic Clearance (CLint) (ml/min/millioncells)=(Vx0.693)/T½; V=incubation volume ml/number of cells.

In Vitro Data

TABLE 4 Cell Growth Inhibition Data MV4; MOL MM. 11 M14 MV4; MOL MV4;MOL 1S SET-2 K562 GI50 GI50 11 M14 11 M14 GI50 CDK8 GI50 GI50 day dayGI50 GI50 GI50 GI50 day Kinase day day 10 10 day 7 day 7 day 3 day 3 10inhibition 10 10 (nM) (nM) (nM) (nM) (nM) (nM) (nM) (100 nM) (nM) (nM)*CA 4 5 9 9 + >1000 racemic- 600 1000 12 racemic- 600 600 17B racemic-150 150 + 13 racemic- 100 40 60 + 16B racemic- 10 20 60 20 40 + 14Bracemic- 10 20 + 14A racemic- 100 40 80 + 15B 14ANO 1.75 14 14B 4 3 87 >1000 14BNO 1 2 >1000 15A 2.5 20 8 15B 12 12.5 >1000 16B 8 9 1517 >1000 17A 1 6 18A 1 3 18B 13 13 20 25 >1000 19A 1.5 5 0.4 19B 8 10 1516 23B 35 55 >1000 24A 1 18 0.5 24B 20 50 26B 4 6 27B 7 10 28B 9.5 1529B 7 14 30B 20 55 31B 75 100 32B 27 55 33B 22 60 34B 8.5 15 35B 80 16036B 19 95 37B 65 200 38B 10 10 39B 17 30 40B 16 25 41B 8 16 42B 7.5 2043A 175 200 43B 95 150 46B ** ** 49B ** ** 50B ** ** 55B ** ** 58B ** **61B ** ** 62B 20 70 62A 0.4 5 0.45 63A 12 30 64A 15 45 65B 3 16 68A 120150 69A 9.5 30 70A 7 12 71B 18 50 71A 2 20 72A 8 4 73A 90 200 >1000 73B150 200 >1000 74A 400 800 >1000 *lack of activity at inhibiting theproliferation of K562 is evidence that analogs are on-target and matchthe selective antiproliferative activity of CA. ** >1,000 nM, day 7.

Other Embodiments

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps.

Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A compound of Formula:

or a pharmaceutically acceptable salt or quaternary amine salt thereof;wherein: m is 0, 1, 2, 3, or 4; R¹ and R² are joined to form a ring offormula:

n is 0, 1, or 2; each instance of R^(6A) is independently halogen, —NO₂,—CN, —OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring. G is —NH—, —NR⁷—, —CH₂—,—CH(R⁷)—, or —C(R⁷)₂—; each instance of R⁷ is independently amino,hydroxyl, halogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, optionally substituted heteroaryl, substituted amino, substitutedhydroxyl, thiol, substituted thiol, carbonyl, sulfonyl, sulfinyl, or anitrogen protecting group when attached to a nitrogen atom; andoptionally wherein two R⁷ groups are joined to form an optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl ring, or an oxo(═O) group;
 2. The compound of claim 1 of Formula:

or a pharmaceutically acceptable salt or quaternary amine salt thereof;wherein: n is 0, 1, or 2; m is 0, 1, 2, 3, or 4; G is —NH—, —NR⁷—,—CH₂—, —CH(R⁷)—, or —C(R⁷)₂—; each instance of R⁷ is independentlyamino, hydroxyl, halogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, substituted amino,substituted hydroxyl, thiol, substituted thiol, carbonyl, sulfonyl,sulfinyl, or a nitrogen protecting group when attached to a nitrogenatom; optionally wherein two R⁷ groups are joined to form an optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl ring, or an oxo(═O) group; each instance of R^(6A) is independently halogen, —NO₂, —CN,—OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; and wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring.
 3. The compound of claim 2,wherein m and n are independently 0, 1, or
 2. 4. The compound of claim3, wherein m is 0 and n is
 1. 5. The compound of claim 4, wherein G is—CH₂—, —CH(R⁷)—, or —C(R⁷)₂—, and each instance of R⁷ is independentlyhydroxyl, amino, or halogen.
 6. The compound of claim 5, wherein G is—CH(R⁷)— and each instance of R⁷ is hydroxyl.
 7. The compound of claim 3wherein m is 0 and n is
 0. 8. The compound of claim 7, wherein G is—CH(R⁷)— and R⁷ is amino.
 9. The compound of claim 1 of Formula:

or a pharmaceutically acceptable salt or quaternary amine salt thereof;wherein: n is 0, 1, or 2; m is 0, 1, 2, 3, or 4; G is —O—, —S—, —NH—,—NR⁷—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—; each instance of R⁷ isindependently amino, hydroxyl, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,substituted amino, substituted hydroxyl, thiol, substituted thiol,carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group whenattached to a nitrogen atom; optionally wherein two R⁷ groups are joinedto form an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl ring, or an oxo (═O) group; each instance of R^(6A) isindependently halogen, —NO₂, —CN, —OR^(6C), SR^(6C), N(R^(6C))₂,—C(═O)R^(6C), —C(═O)OR^(6C), —C(═O)N(R^(6C))₂, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl; andwherein each instance of R^(6C) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached tooxygen, a sulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen, optionally when attached toN the two R^(6C) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring.
 10. The compoundof claim 9, wherein m and n are independently 0, 1, or
 2. 11. Thecompound of claim 10, wherein m and n are
 0. 12. The compound of claim11, wherein G is —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—, and R⁷ is hydroxyl,amino, or halogen.
 13. The compound of claim 12, wherein G is —CH(R⁷)—,and R⁷ is hydroxyl.
 14. The compound of claim 12, wherein G is —CH(R⁷)—,and R⁷ is amino.
 15. The compound of claim 1 of Formula:

or a pharmaceutically acceptable salt or quaternary amine salt thereof;wherein: n is 0, 1, or 2; m is 0, 1, 2, 3, or 4; G is —O—, —S—, —NH—,—NR⁷—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—; each instance of R⁷ isindependently amino, hydroxyl, halogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,substituted amino, substituted hydroxyl, thiol, substituted thiol,carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group whenattached to a nitrogen atom; optionally wherein two R⁷ groups are joinedto form an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl ring, or an oxo (═O) group; each instance of R^(6A) isindependently halogen, —NO₂, —CN, —OR^(6C), SR^(6C), N(R^(6C))₂,—C(═O)R^(6C), —C(═O)OR^(6C), —C(═O)N(R^(6C))₂, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl; andwherein each instance of R^(6C) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, an oxygen protecting group when attached tooxygen, a sulfur protecting group when attached to sulfur, or a nitrogenprotecting group when attached to nitrogen, optionally when attached toN the two R^(6C) groups may be joined to form an optionally substitutedheterocyclyl or optionally substituted heteroaryl ring.
 16. The compoundof claim 15, wherein m and n are independently 0, 1, or
 2. 17. Thecompound of claim 16, wherein G is —O—, —CH₂—, —CH(R⁷)—, or —C(R⁷)₂—,and each instance of R⁷ is independently hydroxyl, amino, or halogen.18. The compound of claim 2, wherein the compound is selected from thegroup consisting of:

and pharmaceutically acceptable salts thereof.
 19. The compound of claim9, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 20. The compound of claim15, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 21. A pharmaceuticalcomposition comprising a compound according to claim 1, or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable excipient.
 22. A method of treating amedical condition mediated by CDK8 and/or CDK19 kinase activity,comprising administering to a subject in need thereof an effectiveamount of a compound according to claim 1, or a pharmaceuticallyacceptable salt, optionally in a pharmaceutical composition.
 23. Themethod of claim 22, wherein the condition is a diabetic condition, aninflammatory condition, macular degeneration, obesity, atherosclerosis,a condition associated with angiogenesis, or a proliferative disorder.24. The method of claim 23, wherein the condition is a proliferativedisorder and the proliferative disorder is primary myelofibrosis (PMF).25. The method of claim 22, wherein the condition is a cancer.
 26. Themethod of claim 25, wherein the cancer is a solid tumor or ahematopoietic cancer.
 27. The method of claim 26, wherein the cancer isa hematopoietic cancer, and the hematopoietic cancer is selected frommultiple myeloma, lymphoma, and acute myelocytic leukemia (AML).
 28. Themethod of claim 22, wherein the subject is a human.
 29. A compound offormula:

or a pharmaceutically acceptable salt thereof; wherein: m is 0, 1, 2, 3,or 4; each instance of R^(6A) is independently halogen, —NO₂, —CN,—OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; and wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring.
 30. A compound of formula:

or a pharmaceutically acceptable salt thereof; wherein: m is 0, 1, 2, 3,or 4; each instance of R^(6A) is independently halogen, —NO₂, —CN,—OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; and wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring.
 31. A compound of formula:

or a pharmaceutically acceptable salt thereof; wherein: m is 0, 1, 2, 3,or 4; R⁴ is hydrogen, halogen, optionally substituted alkyl, or—Si(R^(A))₃; R⁵ is hydrogen, halogen, or optionally substituted alkyl;each instance of

, designated as (a) and (b) represents a single or double bond; R^(O) isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂, oran oxygen protecting group. each instance of R^(A) is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, carbonyl, silyl, an oxygen protectinggroup when attached to oxygen, a sulfur protecting group when attachedto sulfur, or a nitrogen protecting group when attached to nitrogen;optionally when attached to N the two R^(A) groups may be joined to forman optionally substituted heterocyclyl or optionally substitutedheteroaryl ring; each instance of R^(6A) is independently halogen, —NO₂,—CN, —OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; and wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring.
 32. The compound of claim 31,wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 33. A compound offormula:

or a pharmaceutically acceptable salt thereof; wherein: m is 0, 1, 2, 3,or 4; R⁴ is hydrogen, halogen, optionally substituted alkyl, or—Si(R^(A))₃; R⁵ is hydrogen, halogen, or optionally substituted alkyl;each instance of

, designated as (a) and (b) represents a single or double bond; R^(O) isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —C(═O)R^(A), —C(═O)OR^(A), —C(═O)N(R^(A))₂, oran oxygen protecting group. each instance of R^(A) is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, carbonyl, silyl, an oxygen protectinggroup when attached to oxygen, a sulfur protecting group when attachedto sulfur, or a nitrogen protecting group when attached to nitrogen;optionally when attached to N the two R^(A) groups may be joined to forman optionally substituted heterocyclyl or optionally substitutedheteroaryl ring; each instance of R^(6A) is independently halogen, —NO₂,—CN, —OR^(6C), SR^(6C), N(R^(6C))₂, —C(═O)R^(6C), —C(═O)OR^(6C),—C(═O)N(R^(6C))₂, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, or optionally substituted heteroaryl; and wherein each instance ofR^(6C) is independently hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to oxygen, a sulfur protectinggroup when attached to sulfur, or a nitrogen protecting group whenattached to nitrogen, optionally when attached to N the two R^(6C)groups may be joined to form an optionally substituted heterocyclyl oroptionally substituted heteroaryl ring.
 34. A compound of Formula:

or a pharmaceutically acceptable salt thereof.
 35. A pharmaceuticalcomposition comprising compound:

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 36. A method of treating a medical conditionmediated by CDK8 and/or CDK19 kinase activity, comprising administeringto a subject in need thereof an effective amount of compound:

or a pharmaceutically acceptable salt optionally in a pharmaceuticalcomposition thereof.
 37. The method of claim 36, wherein the conditionis a diabetic condition, an inflammatory condition, maculardegeneration, obesity, atherosclerosis, a condition associated withangiogenesis, or a proliferative disorder.
 38. The method of claim 37,wherein the condition is a proliferative disorder and the proliferativedisorder is primary myelofibrosis (PMF).
 39. The method of claim 36,wherein the condition is a cancer.
 40. The method of claim 39, whereinthe cancer is a solid tumor or a hematopoietic cancer.
 41. The method ofclaim 40, wherein the cancer is a hematopoietic cancer, and thehematopoietic cancer is selected from multiple myeloma, lymphoma, andacute myelocytic leukemia (AML).
 42. The method of claim 36, wherein thesubject is a human.
 43. The compound of claim 30, wherein the compoundis:

or a pharmaceutically acceptable salt thereof.